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https://f1000research.com/articles/6-2166/v1	21 Dec 17	{ "type": "Research Article", "title": "Tuition reduction is the key factor determining tax burden of graduate students under the Tax Cuts and Job Act", "authors": [ "Patricia M. Lawston", "Michael T. Parker", "Patricia M. Lawston" ], "abstract": "Background: The proposed Tax Cuts and Jobs Act (H.R.1) has stirred significant public debate on the future of American economics. While supporters of the plan have championed it as a necessity for economic revitalization, detractors have pointed out areas of serious concern, particularly for low- and middle-income Americans. One particularly alarming facet of the plan is the radical change to education finance programs and taxation of students in higher education. Methods: By analyzing actual income and tuition of a public and a private university student, as well as the ‘average’ graduate student, we investigated the effect of both the House and Senate versions of H.R. 1 on taxation of students of various family structures. Results: Our findings indicate that taxable tuition would be the greatest contributor to graduate student tax burden across all four categories of filing status. However, when tuition reduction is upheld or a student is on sustaining fees rather than full tuition, graduate students would realize decreases in taxation. Conclusions: Overall, we conclude that removal of tuition reduction would result in enormous tax burdens for graduate students and their families and that these effects are dependent not only on the status of the student in their degree program but also on their tuition and stipend, and therefore the institution they attend.", "keywords": [ "higher education", "graduate students", "tax reform", "taxes", "tuition reduction" ], "content": "Introduction\n\nAs the Tax Cuts and Jobs Act (H.R.1) inches nearer to becoming law, graduate student concern is mounting to an all-time high. This bill contains changes to how various facets of education are taxed, including student loan interest, education assistance, tax credits, and debt forgiveness1. However, the most jarring change for graduate students is the removal of tuition reduction2, the provision in current tax code allowing students to exclude tuition write-offs from their school from their taxable income. Students are self-reporting that this would increase their yearly taxes between two- and four-fold, resulting in effective tax rates upwards of 33%1,3–7. However, self-reporting can be misleading without in-depth tax calculations included for verification. To address these issues and to help graduate students and elected officials better inform themselves on the impact of proposed tax reform, we analyzed the historical taxes of two graduate students, one public university student and one private university student (the authors of this work). We then extrapolated these analyses for family structures typical of graduate students, including relevant historical and proposed tax deductions and exemptions.\n\n\nMethods\n\nTax calculation notes: Because the Senate version of the Tax Cuts and Jobs act is not yet finalized, we used the “Chairman’s Mark” tax brackets for our calculations8. We excluded the Additional Child Tax Credit from our analyses because this requires more nuanced calculation of tax before it can be determined and is therefore more complex than is appropriate for this simplified, generic example9.\n\nEstimating the ‘average’ graduate student: We estimated the ‘average’ stipend using the current Glassdoor approximation of $30,60310 and then back-calculated for 2013–2015 assuming a 2% yearly increase, as was observed in the public and private student stipends. Tuition was calculated using approximate average public graduate school tuition ($30,000) and average private graduate school tuition ($40,000)11. Then, we weighted the proportion based on an American Academy of Sciences approximation that 60% of PhDs are awarded from public universities12. This gave an average tuition across all students of $34,000 for 2016, which was then back-calculated for 2013–2015 assuming a 2% yearly increase. For health waivers, there is currently no conglomeration of public data available of the average worth provided by universities, so the value used was the average of the public and private student’s actual health waivers ($1,500).\n\nCumulative differential tax burden (CDTB): This value was calculated using the sum of TTO for 2013–2016 under each proposed tax plan (House, House with Tuition Reduction, Senate) minus the sum of TTO for 2013–2016 under historical tax structure. The result is four-year TTO for each filing status (single, single with one child, married, married with one child) under each proposed tax plan.\n\n\nResults\n\nSingle individuals represent the largest category of graduate students (approximately 44.41%13). Table 1 exhibits the calculated taxes for the period 2013–2016 for one individual each from a public and private university using historical tax structure14–19, the House proposed plan20, and the Senate proposed plan8,21. These calculations were made using income, tuition, health waivers, the standard deduction (typical for graduate students), and deductions and exemptions that are germane to this group. Representation of the total taxes owed (TTO) and effective tax rates (ETR = TTO / stipend) are shown in Table 1, showing maximum increases under the House plan of approximately +386% and +19%, respectively. For the public student, this jump in both categories can partly be attributed to a low initial tax burden that is substantially increased when tuition is considered taxable income. The increases are more modest in years where this student was on sustaining fees (2015 and 2016) rather than full tuition. When considering the Senate plan, which does not tax tuition22, there are actually large decreases in TTO and modest decreases in ETR (maximum of approximately -29% and -1.75%, respectively). Interestingly, when considering the House plan without taxable tuition, the TTO and ETR both decrease as well (maximum of approximately -14.5% and -1.0%, respectively). This highlights the extreme effect this one change would have on graduate student tax burden.\n\nstudent on sustaining status\n\nThe results for the student from a private university are similar to those above (Table 1). The House plan results in approximate maximum increases of +192% TTO and +19% ETR, while both the House plan that retains tuition reduction and the Senate plan result in decreased TTO and ETR (approximate maximum of -18.5% TTO and -1.75% ETR for the House plan without taxable tuition; -24% TTO and -2.5% ETR for Senate plan). This student also went from full tuition to sustaining between 2015 and 2016, explaining the modest decrease in TTO and ETR.\n\nFigure 1 shows, in U.S. Dollars (USD), the TTO for single individuals from a public (A) and a private (B) university based upon the type of tax plan applied. For the public student, TTO increases under the House plan, and decreases under the House plan with tuition reduction and the Senate plan (Figure 1A). When tuition is taxed and the student is on full tuition, the TTO increases from $872 to $4238 in 2013 and from $907 to $4380 in 2014. For the private school student, who was on full tuition from 2013–2015, the increases in TTO under the House plan are $3,388 to $9,550 in 2013, $3,574 to $10,201 in 2014, and $3,649 to $10,669 in 2015 (Figure 1B). Also, similar to the public student calculations, when the private student transitioned to sustaining fees in 2016, they would realize a decreased TTO under the House plan (-$213), although this decrease would be even greater without taxable tuition and with the Senate plan (-$623 and -$813, respectively). Overall, these analyses indicate that the proposed tax plans would generally decrease a single graduate student’s tax burden unless tuition is treated as taxable income, in which case there would be huge increases in TTO.\n\nTotal Taxes Owed (TTO) represented in USD for single filing public (A) and private (B) university graduate students. TTO for each year (2013–2016) are color coded by year and grouped according to the tax structure utilized to make each calculation (Table 1). These structures were the historical taxation structures for years 2013–2016, the proposed House tax plan, the proposed House plan with Tuition Reduction, and the proposed Senate tax plan. Above each bar is the calculated Effective Tax Rate (ETR) for each scenario, represented as a percent of income.\n\nThe changing status and income of graduate students over their programs and the effect this has on taxation is worth noting. Students go on and off of fellowships, affecting the amount and duration of their pay. The public student’s stipend was guaranteed for nine months of the year, but when funded by an outside (e.g., NSF) fellowship, the stipend was year-long, leading to greater annual income when on fellowship. And in later years of their degree, many students no longer have traditional tuition costs, but rather pay sustaining fees, greatly reducing their effective income under the House plan. In Figure 1A, the TTO of the public student responds to these fluctuating statuses under each plan. In 2013 and 2014, this student was off of fellowship and on full tuition, then went off of full tuition for 2015 and 2016, and in 2016 was on fellowship. Between 2014 and 2015, the transition to sustaining fees has little effect on plans that do not tax tuition, but under the House plan there is a large decrease (-$3,168), though this plan still results in an increase as compared to the others (+$260 vs. historical taxation). This highlights that sustaining fee status greatly reduces the effect of the House tax plan on graduate student tax increases. When the student goes onto fellowship in 2016 their stipend increases, with a concomitant increase in taxation. However, the increase in taxes under the House plan vs. the historical structure remains low (+$199), exhibiting that an increase in stipend (~30% in this case) has minimal effect on taxation as compared to that of taxing tuition.\n\nWhile the above data represent the major population of graduate students, they do not paint the whole picture of tax scenarios as a graduate student. We identified three other common family structures of graduate students and calculated taxes for each based on historical and proposed tax policies. These groups and their proportion of the graduate student population were: Single with dependents (12.30%), married without dependents (14.95%), and married with dependents (28.34%)13. It is important to mention that in keeping with the subject of this study, namely, graduate students, married couple calculations assume that both partners are students at the same university, and therefore the income, tuition, and health waivers are double that of a single student. Another factor that we controlled for simplicity of calculations was to assume that families that have children only had one.\n\nTable 2 contains the tax calculations for each of the abovementioned groups using historical and proposed tax structures. Similar to single filers, the House plan would increase graduate student taxes across the board when tuition is considered as taxable. For the public student, TTO and ETR would increase an approximate maximum of +866% / +9.5% as a single filer with one child, +188% / +8.8% as a married couple filing jointly, and +385% / +5.7% as a married couple filing jointly with one child. In all cases, when tuition is not taxed in proposed plans, decreases in TTO and ETR are observed as compared to historical tax structures. Approximate maximum decreases in TTO and ETR under the Senate plan are -29% / -1.8% for Married Filing Jointly (MFJ) and -29% / -4.5% for MFJ with one child. The House plan without taxable tuition has similar, albeit more modest decreases in TTO and ETR (MFJ = -14.5% / -1.1%; MFJ with one child = -14.5% / -3.7%). Strikingly, the single filer with one child would actually owe no taxes to the government for any year if tuition is not taxable.\n\nstudent on sustaining status\n\nstudent on sustaining status\n\nstudent on sustaining status\n\nThe private student would encounter similar tax scenarios, with approximate maximum increases in TTO and ETR under the House plan of +443% / +16.3% as a single with one child, +52.4% / +5.1% as MFJ, and +67.1% / +5.07% as MFJ with one child. Removal of taxable tuition results in decreased taxation in all cases, with the House plan performing better for singles with one child (-63.25% / -2.15%) and the Senate plan performing better for married filers (MFJ = -24% / -1.75%; MFJ with one child = -39.25% / -1.8%).\n\nFigure 2 shows the range of TTO in USD for the various family structures of graduate students from a public (A) and a private (B) university based upon the type of tax plan applied. When tuition is taxed under the House plan, TTO increases in all cases for all filers. For the public student these increases were greatest with 2014 data (single with one child = $222 to 2,048; MFJ = $1,814 to $5,187; MFJ with one child = $1,419 to $3,587), the last year this student was on full tuition (Figure 2A). When calculations were made using the House plan without taxable tuition and/or the Senate plan, TTO decreased in all cases. And when the student was no longer on full tuition (2015 and 2016) TTO actually decreased when comparing the House plan with the historical structure in all cases (with the exception of MFJ in 2015). In 2015, these changes were: Single = -$260; single with one child = -$247; MFJ = +$174; MFJ with one child = -$1,006. In 2016, these changes were: Single = -$199; single with one child = -$927; MFJ = -$92; MFJ with one child = -$1,085.\n\nTotal Taxes Owed (TTO) represented in USD for public (A) and private (B) university graduate students filing either as single with one child, married (filing jointly), or married with one child (filing jointly). TTO for each year (2013–2016) are color coded by year and grouped according to the tax structure utilized to make each calculation (Table 2). These structures were the historical taxation structures for years 2013–2016, the proposed House tax plan, the proposed House plan with Tuition Reduction, and the proposed Senate tax plan. A “0” represents no taxes owed.\n\nThe increase in TTO was also largest for the private school student in the last year of full tuition, 2015 (single with one child = $1,410 to 7,544; MFJ = $7,298 to $11,119; MFJ with one child = $5,698 to $9,519) (Figure 2B). For all family types and in all cases, when the private school student was no longer on full tuition or when calculations were made using proposed plans without taxable tuition, there were decreases in TTO, though these were most striking for couples (ex.: 2016, MFJ with one child: Historical = $5,895; House = $5,038, [-$857]; House without taxable tuition = $4,628, [-$1,267]; Senate = $3,847, [-$2,048]). Overall, these analyses indicate that the proposed tax plans would generally decrease graduate student family tax burden unless tuition is treated as taxable income. Both new plans benefit students in the form of decreased taxation when they are on sustaining fees near the end of their degrees. Additionally, the Senate plan results in more tax relief than any other tax structure for all family types.\n\nWhile utilizing taxes from representative students is useful to delineate between type of institution, it is more generally applicable to analyze the average income, tuition, and health waiver of a typical graduate student. These calculations (Dataset 3) follow the same trends as those for the individuals above, and the effect of various tax plans on TTO are shown below in Figure 3. Increases in TTO under the House plan were most drastic using 2016 estimates (single = $2,566 to $7,550; single with one child = $320 to $4,425; MFJ = $5,148 to $8,604; MFJ with one child = $3,540 to $7,004). As before, removal of taxable tuition from the House plan and/or the Senate plan results in overall decreases in TTO (Example, 2016, single: Historical = $2,566; House without taxable tuition = $2,172, [-$394]; Senate = $1,982, [-$584]).\n\nTotal Taxes Owed (TTO) represented in USD for an ‘average’ university graduate student filing either as single, single with one child, married (filing jointly), or married with one child (filing jointly). TTO for each year (2013–2016) are color coded by year and grouped according to the tax structure utilized to make each calculation (Datasets 1–3, Tables S1–S6). These structures were the historical taxation structures for years 2013–2016, the proposed House tax plan, the proposed House plan with Tuition Reduction, and the proposed Senate tax plan. A “0” represents no taxes owed.\n\nThe cumulative effect of tax reform on graduate students is likely the most informative measure of potential impacts. In Figure 4, we calculate the cumulative difference in taxes over four years with both former and proposed tax plans for the public student (A), private student (B), and average student (C). For the public student, the cumulative differential tax burden (CDTB, see Methods) over four years under the House tax plan is $7,297 for a single filer and $2,107 for a single with one child (Figure 4A). These figures are marginally lower for married filers ($6,695 to $2,361), as expected. These results highlight that, under the House plan, dependent child status has a greater buffering impact than does marriage. For the private student, whose stipend and tuition are greater than the public student, the CDTB values are: Single = $19,597; single with one child = $16,795; MFJ = $9,837; MFJ with one child = $9,822 (Figure 4B). For single and single with one child, these tax increases are equal to approximately one-eighth the student’s total stipend income over four years. Interestingly, at higher income and tuition marriage status is the larger buffer, apparently due to a low initial tax burden for those with children at lower incomes. This also proves true for the CDTB of the average graduate student: Single = $18,451; single with one child = $14,941; MFJ = $13,325; MFJ with one child = $13,310 (Figure 4C). Strikingly, the CDTB is always negative when tuition is not taxable, meaning that the new tax plans would reduce cumulative taxation when compared to the historical structure. These data indicate that graduate students would actually benefit from the House and Senate tax plans in the form of decreased tax burden, as long as tuition is not taxable.\n\nCumulative Differential Tax Burden (CDTB) represented in USD for a public university (A), private university (B), or ‘average’ (C) graduate student. CDTB is the TTO for four years (2013–2016) and is coded by filing status (single, single with one child, married [filing jointly], or married with one child [filing jointly]) and grouped according to the tax structure utilized to make each calculation (Datasets 1–3, Tables S1–S6). These structures were the historical taxation structures for years 2013–2016, the proposed House tax plan, the proposed House plan with Tuition Reduction, and the proposed Senate tax plan.\n\nAs has been alluded to throughout this work, it would appear that taxation of tuition is the single most important factor affecting graduate students in the House tax plan. In Figure 5, we show the effect of including tuition reduction in the House tax plan for one tax year, using 2016 estimations. For all graduate student family structures, changing this single factor immensely decreases the tax burden in all cases, anywhere from -$4,260 (MFJ with one child) to -$5,378 (single). This is a larger decrease than is realized for marriage (-$3,248 per person), having a child (-$3,125), or even the combination of these two (-$4,048 per person).\n\nTotal Taxes Owed (TTO) in 2016, represented in USD, for an ‘average’ university graduate student filing either as single, single with one child, married (filing jointly), or married with one child (filing jointly). Data are coded according to the tax structure utilized to make each calculation (Datasets 1–3, Tables S1–S6). These structures were the proposed House tax plan and the proposed House plan with Tuition Reduction. A “0” represents no taxes owed.\n\n\nDiscussion\n\nIn this work, it was our goal to better understand the effects of the proposed Tax Cuts and Jobs Act on graduate students and to provide accurate information to help students, their supporters, and elected officials make informed decisions in their stand on the matter. Our findings indicate that taxable tuition would be the greatest contributor to graduate student tax burden across all four categories of filing status. However, when tuition is removed from the equation (whether in our modified House plan, the Senate plan, or by transition of students to sustaining fees) there is a decrease in tax burden in all situations. This gives validity to claims that these new tax plans would generally benefit low and middle-income families in the form of reduced taxation, at least in the short term23,24. But for graduate students, it would appear that these benefits would not be realized if tuition is considered taxable income.\n\nOverall, we conclude that exclusion of tuition reduction from H.R.1, or for that matter any future iteration of U.S. Tax Code, would be an enormous financial burden on graduate students. What this would mean for graduate education in the US is uncertain, but will likely impact what schools will be able to host graduate programs, who can afford graduate education, and the diversity of the students within graduate programs.\n\n\nData availability\n\nData for the two graduate student tuitions, stipends, and health waivers were provided directly from the authors from their personal tax information. These values can be found in the respective columns in Dataset 1 and Dataset 2.\n\nValues used to calculate the ‘average’ graduate student were obtained via Glassdoor10, MoneyUnder3011, and the American Academy of Sciences12 (see Methods) or estimated from the mean of the two student values available in the absence of available data on health waivers and can be found in Dataset 3. All data is publicly available.\n\nDataset 1. Calculation of 2013–2016 taxes for a public university graduate student using historical tax plans. DOI, http://dx.doi.org/10.5256/f1000research.13385.d18815625\n\nDataset 2. Calculation of 2013–2016 taxes for a private university graduate student using historical tax plans. DOI, http://dx.doi.org/10.5256/f1000research.13385.d18815726\n\nDataset 3. Calculation of 2013–2016 taxes for the ‘average’ university graduate student using historical tax plans. DOI, http://dx.doi.org/10.5256/f1000research.13385.d18815827", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nTable S1. Calculation of 2013–2016 taxes for a public university graduate student using the proposed House tax plan, with and without tuition reduction.\n\nClick here to access the data.\n\nTable S2. Calculation of 2013–2016 taxes for a private university graduate student using the proposed House tax plan, with and without tuition reduction.\n\nClick here to access the data.\n\nTable S3. Calculation of 2013–2016 taxes for the ‘average’ university graduate student using the proposed House tax plan, with and without tuition reduction.\n\nClick here to access the data.\n\nTable S4. Calculation of 2013–2016 taxes for a public university graduate student using the proposed Senate tax plan.\n\nClick here to access the data.\n\nTable S5. Calculation of 2013–2016 taxes for a private university graduate student using the proposed Senate tax plan.\n\nClick here to access the data.\n\nTable S6. Calculation of 2013–2016 taxes for the ‘average’ university graduate student using the proposed Senate tax plan.\n\nClick here to access the data.\n\n\nReferences\n\nFigueroa A: Here’s How The New Tax Plan Could Hurt Graduate Students. NPR. 2017. Reference Source\n\nWould the Republican Tax Plan Count Graduate Tuition Waivers as Taxable Income? Snopes. 2017. Reference Source\n\nWalsh D: I’m a grad student, and the Republican tax plan could cost me thousands of dollars. The Washington Post. 2017. Reference Source\n\nGonzalez R: Grad Students are Freaking Out About the GOP Tax Plan. They Should Be. WIRED. 2017. Reference Source\n\nReilly K: Graduate Students Sound Alarm on Huge Increases Under GOP Tax Plan: ‘I Would Be Hopelessly Priced Out’. Time. 2017. Reference Source\n\nRousseau E: The House Just Voted to Bankrupt Graduate Students. The New York Times. 2017. Reference Source\n\nGraduate students face alarming tax hike. Nature. 2017. Reference Source\n\nKaeding N: Overview of the Senate’s Amendment to the Tax Cuts and Jobs Act. Tax Foundation. 2017. Reference Source\n\nWhat is the Additional Child Tax Credit? TurboTax. 2017. Reference Source\n\nGraduate Student Salaries. Glassdoor. 2017. Reference Source\n\nBarret L: Grad School ROI Calculator: Is Graduate School Worth The Cost? MoneyUnder30. 2015. Reference Source\n\nAmerican Academy of Arts & Sciences: Public Research Universities: Why They Matter. 2015. Reference Source\n\nNCES: Number and percentage of graduate students taking night, weekend, or online classes, by selected characteristics: 2011–12. Digest of Education Statistics. 2013. Reference Source\n\nKasprak N: 2013 Tax Brackets. Tax Foundation. 2013. Reference Source\n\nErb KP: IRS Announces 2013 Tax Rates, Standard Deduction Amounts And More. Forbes. 2013. Reference Source\n\nPomerleau K: 2014 Tax Brackets. Tax Foundation. 2013. Reference Source\n\nPomerleau K: 2015 Tax Brackets. Tax Foundation. 2014. Reference Source\n\n26 U.S.C. § 24. (Congress, 2014).\n\nPomerleau K: 2016 Tax Brackets. Tax Foundation. 2015. Reference Source\n\nUpdated Details and Analysis of the 2017 House Tax Cuts and Jobs Act. Tax Foundation. 2017. Reference Source\n\nTax Cuts & Jobs Act \| Senate Tax Reform Bill. National Council of Nonprofits. 2017. Reference Source\n\nDouglas-Gabriel D: Endowment tax remains in play, but graduate tax is off the table in Senate plan. The Washington Post. 2017. Reference Source\n\nGOP: WTAS: Trump’s Tax Cuts & Jobs Act. Press Releases. 2017. Reference Source\n\nPaletta D, DeBonis M: GOP tax plan would shrink mortgage interest benefit, slash corporate tax rate. The Washington Post. 2017. Reference Source\n\nLawston P, Parker M: Dataset 1 in: Tuition reduction is the key factor determining tax burden of graduate students under the Tax Cuts and Job Act. F1000Research. 2017. Data Source\n\nLawston P, Parker M: Dataset 2 in: Tuition reduction is the key factor determining tax burden of graduate students under the Tax Cuts and Job Act. F1000Research. 2017. Data Source\n\nLawston P, Parker M: Dataset 3 in: Tuition reduction is the key factor determining tax burden of graduate students under the Tax Cuts and Job Act. F1000Research. 2017. Data Source" }	[ { "id": "29597", "date": "23 Jan 2018", "name": "Joshua D Hall", "expertise": [ "Reviewer Expertise Biomedical graduate education", "microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis well-written article examines the financial impact that changes to the US tax code would have on graduate students who receive paid tuition as a benefit. This is a timely and important topic being discussed by graduate students and those involved with graduate education across the country. While the final, passed tax bill did not include taxation of graduate student tuition benefits, this study will provide useful data for demonstrating the significant financial burden such a change would levy upon graduate students if it comes up for discussion in the future.\nA few specific comments and suggestions.\nAs a technical point, the term “tuition reduction” is used to describe the benefit that graduate students would potentially no longer be able to deduct. However, in biomedical graduate programs that I am familiar with, the grad student tuition is not waived or reduced, but paid on behalf of the graduate student by their department or thesis lab. This may be what the authors meant by this term, but this should be clarified to avoid confusion. For estimation of “average” graduate student salaries, it is unclear if this is for graduate students of all disciplines or for students from only STEM disciplines, which tend to have higher stipend levels. The $30,603 average seems high for an average inclusive of all graduate students independent of discipline. Please define “TTO” the first time it is used. I’m not familiar with the term “sustaining fees”. I’m deducing that this means the student did not have to pay tuition for that year for some reason, but this should be defined explicitly in the text, especially because it has a big influence on the results in Table/Figure 1. While individual funding structures for graduate students can vary widely from discipline to discipline (and even year to year as the data from Fig 1 indicates for individual students), the analysis in Figure 3 is particularly useful for showing the taxation impact on graduate students overall as a result of a taxation plan that includes taxation of tuition benefits.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3396", "date": "05 Feb 2018", "name": "Michael Parker", "role": "Author Response", "response": "Thank you for your time in reviewing our manuscript and for your insight and suggestions. We address each by the number system you utilized for commenting and hope that the edits in Version 2 meet your expectations. Reviewer comment 1:As a technical point, the term “tuition reduction” is used to describe the benefit that graduate students would potentially no longer be able to deduct. However, in biomedical graduate programs that I am familiar with, the grad student tuition is not waived or reduced, but paid on behalf of the graduate student by their department or thesis lab. This may be what the authors meant by this term, but this should be clarified to avoid confusion. Authors response 1.Thanks for pointing this out. We edited the wording in the introduction to make it clearer that this typically refers to tuition paid for the student by department money. This section now reads:“However, the most jarring change for graduate students is the removal of tuition reduction 2 , the provision in current tax code allowing students to exclude money provided by their graduate program to cover their tuition costs from their taxable income.” Reviewer comment 2: For estimation of “average” graduate student salaries, it is unclear if this is for graduate students of all disciplines or for students from only STEM disciplines, which tend to have higher stipend levels. The $30,603 average seems high for an average inclusive of all graduate students independent of discipline. Authors response 2: Although we agree that this average seems high, it is the best approximation we can make from anonymous self-reporting. It would appear that the Glassdoor site may have a bias toward STEM fields, but it is not clear. We provided a statement in the methods to note this uncertainty, which hopefully provides more clarity on the nature of the approximation. This revised section reads as follows: “We estimated the ‘average’ stipend using the current Glassdoor approximation of $30,603 10 and then back-calculated for 2013–2015 assuming a 2% yearly increase, as was observed in the public and private student stipends. One consideration in this approximation is that these data are self-reported and not field specified and since STEM fields typically have higher stipends than humanities, bias in either discipline’s direction may skew this value.” Reviewer comment 3: Please define “TTO” the first time it is used. Authors’ response 3:Done. Additionally, we have added an additional section describing TTO more fully to the methods to address both reviewer comments about TTO. Reviewer comment 4:I’m not familiar with the term “sustaining fees”. I’m deducing that this means the student did not have to pay tuition for that year for some reason, but this should be defined explicitly in the text, especially because it has a big influence on the results in Table/Figure 1. Authors’ response 4:Thanks for calling this to our attention. Students are typically placed on sustaining status after they have completed their coursework, but still have other degree requirements to fill. This is most often research and/or the completion of a thesis. Tuition is not charged since the student is not enrolled in classes. Instead a sustaining fee is charged in exchange for the student’s active status and to allow continued use of the University resources. We have revised the Methods section to include an additional paragraph which describes the sustaining fees/status. It now reads as follows:“Sustaining fees: Once graduate students progress from the class and teaching portions of their graduate work into the research intensive/exclusive portion, many schools no longer charge full tuition, but rather a smaller fee termed a “sustaining fee”. In the case of both graduate student salaries utilized in this work, this was the case, and the sustaining fees for each can be found in Datasets 1 and 2. The public student was on sustaining status in 2015 and 2016 while the private student was on sustaining status in 2016. The reduction in taxation is drastic when students transition from tuition status to sustaining status under plans that do not contain the tuition reduction provision (see Figures 1 and 2).” Reviewer comment 5:While individual funding structures for graduate students can vary widely from discipline to discipline (and even year to year as the data from Fig 1 indicates for individual students), the analysis in Figure 3 is particularly useful for showing the taxation impact on graduate students overall as a result of a taxation plan that includes taxation of tuition benefits. Authors’ response 5:Thank you for your kind words and for your support of the reasoning behind this figure. We, too, believe that these data are important and hope they will be widely useful for readers of our manuscript." } ] }, { "id": "29283", "date": "30 Jan 2018", "name": "Elizabeth Garrett-Mayer", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nPage 3: What is TTO? Not defined prior to its introduction in the Methods. It is defined in the Results instead.\n\nAssumptions regarding income, health waivers and stipends are not well-described. In table 1, what income level is this based on? In the results, it is stated that “this student” is on sustaining fees and not full tuition. How would one know that? The assumptions should be plainly described in the methods section AND in the caption of the table. It seems that the authors are actually following the tax burden of a single graduate student where each year (2013-2016) represents that students year in graduate school. This is not clear at all. The methods need to be substantially improved.\n\nThe increase to 386% is misleading. It would seem relevant to calculate change from the most recent year of data, not 2013. The “sustaining” status is not clearly defined. This is obviously critically important. In Table 1, why does 2015 have an asterisk in the top row but not other rows? It would seem more logical and less confusing to take a single year (e.g. 2016) and compare the tax implications for students in each of the different settings. That is, showing the 2016 tax burden under each plan for students with full tuition, sustaining fees, stipend, different levels of other income or filing status, etc. The way it is now, because the methods do not emphasize (nor do most of the figures), it looks like 2013 is very odd.\n\nWhy does that stipend double when filing jointly?\n\nThere is no actual ‘data’, but the author provide the numbers on which they based their assumptions. I have serious concerns that the results will be misinterpreted due to the presentation of results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3395", "date": "05 Feb 2018", "name": "Michael Parker", "role": "Author Response", "response": "Thank you for your time in reviewing our manuscript and for your insight and suggestions. We address each comment and suggestion in the order presented and hope that the edits in Version 2 meet your expectations. The original comments are shown in bold type font and our responses in regular font. Where changes have been made to the manuscript, the relevant text has been copied and pasted here and is shown within quotation marks. Reviewer comment 1: Page 3: What is TTO? Not defined prior to its introduction in the Methods. It is defined in the Results instead. Authors response 1: We have added a paragraph devoted to TTO in the Methods section. It now reads as follows: “Total taxes owed (TTO): For each year 2013–2016, the appropriate tax brackets were applied to calculate the total taxes owed (TTO) based on stipends, health waivers, and tuition. Representation of TTO is calculated by year, and differences in TTO for each proposed tax structure are in relation to that same year’s historical tax structure TTO calculation. The income values used for these calculations as well as the numerous factors considered in tax calculations (i.e., column headers) can all be found in Datasets 1–3.” Reviewer comment 2a: Assumptions regarding income, health waivers and stipends are not well-described. Authors response 2a: To more fully describe the income, health waiver, and stipend, additional information was added to the new TTO paragraph in the Methods as cited above. For the single public and private university students, no assumptions are made; rather the self-reported data by each student is used as provided. Assumptions for the ‘average’ grad student are given under the title subtitle ‘Estimating the average graduate student’ in the Methods section. Reviewer comment 2b: In table 1, what income level is this based on? Authors response 2b: For the Historical, House w/tuition reduction, and Senate scenarios, the income level used to calculate the tax burden in Table 1 is the student stipend only. Under the House plan, the income level is the sum of the stipend, health waiver, and tuition amounts as all three would be considered taxable income. The supplementary tables, S1-S6, include the exact stipend, health waiver, and tuition amounts for each student and each year. Reviewer comment 2c: In the results, it is stated that “this student” is on sustaining fees and not full tuition. How would one know that? The assumptions should be plainly described in the methods section AND in the caption of the table. It seems that the authors are actually following the tax burden of a single graduate student where each year (2013-2016) represents that students year in graduate school. This is not clear at all. The methods need to be substantially improved. Authors response 2c: An additional paragraph has been added to the methods section detailing the unique position of sustaining status. It reads as follows: “Sustaining fees: Once graduate students progress from the class and teaching portions of their graduate work into the research intensive/exclusive portion, many schools no longer charge full tuition, but rather a smaller fee termed a “sustaining fee”. In the case of both graduate student salaries utilized in this work, this was the case, and the sustaining fees for each can be found in Datasets 1 and 2. The public student was on sustaining status in 2015 and 2016 while the private student was on sustaining status in 2016. The reduction in taxation is drastic when students transition from tuition status to sustaining status under plans that do not contain the tuition reduction provision (see Figures 1 and 2).” In addition, the introduction section has been revised to more clearly state that the years cited are in fact the progression of each graduate student through PhD programs: “To address these issues and to help graduate students and elected officials better inform themselves on the impact of proposed tax reform, we analyzed the historical taxes of two graduate students, one public university student and one private university student (the authors of this work), over the course of four years (2013-2016) in science PhD programs.\" Additionally, the caption of Table 1 had been revised as suggested to note the sustaining status and that the numbers can be found in the supplementary tables. Reviewer comment 3a: The increase to 386% is misleading. It would seem relevant to calculate change from the most recent year of data, not 2013. Author response 3a: The ‘historical’ taxes are those that have been paid by each student in each year of graduate study (2013-2016), and serves essentially as the ‘control’ case. Thus, the differences (both absolute and percent change) use the ‘historical’ tax owed in the respective year as the reference value when calculating the changes under each plan. For example, the public student filing singly in 2013 owed $872, as the student’s stipend (and therefore gross income) was $18,724. If the House plan had been in effect at that time, the student’s gross income would have instead been the sum of the student’s stipend, health waiver ($1,273), and tuition waiver ($27,822), bringing the student’s gross income for 2013 to $47,819. Therefore, the student would have owed $4,238 in taxes in 2013, if the House plan had been in effect; an increase of 385% over the historical tax burden. We use this retrospective analysis approach to quantify the potential each of the proposed plans on graduate student tax burden. Reviewer comment 3b: The “sustaining” status is not clearly defined. This is obviously critically important. In Table 1, why does 2015 have an asterisk in the top row but not other rows? Authors’ response 3b: Thank you for calling the ambiguity related to sustaining fees to our attention, which was echoed by the second reviewer. As a result of these comments, we’ve revised the Methods section to include an additional paragraph which describes the sustaining fees/status. It now reads as follows: “Sustaining fees: Once graduate students progress from the class and teaching portions of their graduate work into the research intensive/exclusive portion, many schools no longer charge full tuition, but rather a smaller fee termed a “sustaining fee”. In the case of both graduate student salaries utilized in this work, this was the case, and the sustaining fees for each can be found in Datasets 1 and 2. The public student was on sustaining status in 2015 and 2016 while the private student was on sustaining status in 2016. The reduction in taxation is drastic when students transition from tuition status to sustaining status under plans that do not contain the tuition reduction provision (see Figures 1 and 2).” With respect to the asterisk in the top row, this was supposed to be included for the 2015 headers for the public student but erroneously excluded. Thank you for bringing it to our attention. We have updated Table 1 to fix this issue. Reviewer comment 3c: It would seem more logical and less confusing to take a single year (e.g. 2016) and compare the tax implications for students in each of the different settings. That is, showing the 2016 tax burden under each plan for students with full tuition, sustaining fees, stipend, different levels of other income or filing status, etc. The way it is now, because the methods do not emphasize (nor do most of the figures), it looks like 2013 is very odd. We agree that presenting the data in the way suggested would more clearly display the impacts of each plan on the respective categories listed. However, a main goal of this work was to better understand the cumulative impacts of these potential plans over the course of a graduate student’s career; a career that is not typically not limited to any one of these settings (e.g., full tuition, sustaining, etc.) but rather is an aggregate of each of these and other factors that change yearly. Thus, we feel it provides a distinct advantage and is more representative to display the data in the way chosen. For example, Fig. 1 shows that despite the yearly variability in taxes owed, especially for the public university student (due to a 2016 increased stipend), the House plan greatly increases the tax burden for both students. However, the House plan with the tuition reduction maintained and the Senate plan both decrease the tax burden for both students. Reviewer comment 4: Why does that stipend double when filing jointly? Although the majority of graduate students are unmarried, we acknowledge that other family structures exist and these structures could be particularly vulnerable to drastic changes in the tax code. Thus, we extended the analysis to consider single graduate students with one child, married graduate students, and married graduate students with one child. As this analysis is centered specifically on graduate students, we consider only marriages where the couple is comprised of two graduate students at the same university. As a result, the income for the couple is defined as twice the individual student’s yearly stipend. We acknowledge that a married graduate student could be married to any variety of partners, and thus why we limit the analysis to only graduate student pairs. This is described in paragraph 5 of the results: “It is important to mention that in keeping with the subject of this study, namely, graduate students, married couple calculations assume that both partners are students at the same university, and therefore the income, tuition, and health waivers are double that of a single student.\" Reviewer comment 5: There is no actual ‘data’, but the author provide the numbers on which they based their assumptions. I have serious concerns that the results will be misinterpreted due to the presentation of results. Author response 5: The data provided are the actual student stipends, health waiver amounts, and tuition costs for four years for two graduate students – one at a public university and one at a private university. These data are taken directly from pay stubs and student financial statements willingly disclosed by the students; the authors of this manuscript. Although it would be ideal to have a large sample size of student financial information, we feel it is not necessary for the scope of this work. As public universities tend to offer lower stipends and private universities typically the highest, the public v. private university contrast offers lower and upper bounds on the potential impacts of each of these plans. This is confirmed by the information obtained from Glassdoor that shows the average stipend for a graduate student falls between that reported by the public and private university students. We hope that the explanations provided in these responses and the changes to the manuscript made as a result of the thoughtful reviewer comments have helped to clarify the data and assumptions and have generally improved the presentation of the manuscript." } ] } ]	1	https://f1000research.com/articles/6-2166
https://f1000research.com/articles/6-1787/v1	02 Oct 17	{ "type": "Opinion Article", "title": "The sedentary (r)evolution: Have we lost our metabolic flexibility?", "authors": [ "Jens Freese", "Rainer Johannes Klement", "Begoña Ruiz-Núñez", "Sebastian Schwarz", "Helmut Lötzerich", "Rainer Johannes Klement", "Begoña Ruiz-Núñez", "Sebastian Schwarz", "Helmut Lötzerich" ], "abstract": "During the course of evolution, up until the agricultural revolution, environmental fluctuations forced the human species to develop a flexible metabolism in order to adapt its energy needs to various climate, seasonal and vegetation conditions. Metabolic flexibility safeguarded human survival independent of food availability. In modern times, humans switched their primal lifestyle towards a constant availability of energy-dense, yet often nutrient-deficient, foods, persistent psycho-emotional stressors and a lack of exercise. As a result, humans progressively gain metabolic disorders, such as the metabolic syndrome, type 2 diabetes, non-alcoholic fatty liver disease, certain types of cancer, cardiovascular disease and Alzheimer´s disease, wherever the sedentary lifestyle spreads in the world. For more than 2.5 million years, our capability to store fat for times of food shortage was an outstanding survival advantage. Nowadays, the same survival strategy in a completely altered surrounding is responsible for a constant accumulation of body fat. In this article, we argue that the metabolic epidemic is largely based on a deficit in metabolic flexibility. We hypothesize that the modern energetic inflexibility, typically displayed by symptoms of neuroglycopenia, can be reversed by re-cultivating suppressed metabolic programs, which became obsolete in an affluent environment, particularly the ability to easily switch to ketone body and fat oxidation. In a simplified model, the basic metabolic programs of humans’ primal hunter-gatherer lifestyle are opposed to the current sedentary lifestyle. Those metabolic programs, which are chronically neglected in modern surroundings, are identified and conclusions for the prevention of chronic metabolic diseases are drawn.", "keywords": [ "Western diseases", "Evolution", "Hunter-Gatherer Lifestyle", "Sedentary Lifestyle", "Metabolic flexibility" ], "content": "\n\nCharles Darwin: “It is not the strongest of the species that survives, nor the most intelligent, but the one most responsive to change.”\n\n\nIntroduction\n\nDuring the longest time of Homo sapiens’ existence, approximately 99.5% or 84.000 generations1–3, humans’ daily survival was shaped by food insecurity (feast and famine), adaptation to a widespread range of different food sources (biodiversity), abundant daily exercise frequently under fasting conditions (foraging behavior), as well as unpredictable food supply (intermitted fasting), depending on the daily foraging success. Consequently, in the course of evolution, environmental stress forced the human species to develop an extraordinary flexible metabolism with multiple programs to guarantee energy equilibrium. Lipolysis, proteolysis, gluconeogenesis, ketone bodies and muscle-derived lactate as alternative energy substrates for cerebral neurons, provide examples by which natural selection tailored humans to buffer fluctuations in energy supply4.\n\nUntil the Agricultural revolution around 12.000-10.000 BC, when humans began to store food on a larger scale for periods of shortage, survival would have depended on the adaption of energy needs to various climate, seasonal and vegetation conditions. Metabolic flexibility, optimized in millions of years of adaptation to environmental stimuli, safeguarded human survival independent of food disposability5,6. Due to an overflow of energy in modern times, humans have inevitably modified many evolutionary-based behavior patterns. As an example, when the body is threatened by energy depletion, hunger normally activates foraging behavior as a prioritized drive in both wild animals and hunter-gatherers, in order to anticipate new food resources7,8. Nowadays, hunting and gathering need only a few steps into the kitchen or a short drive to the local food store, which implies substantial energy intake by no or less energy expenditure in the locomotor system9–11.\n\nIn an evolutionary context, humans switched their primal lifestyle towards a plethora of energy-dense foods, persistent psycho-emotional stressors and a dramatic lack of exercise in record time. As a result of this ultra-rapid metamorphosis, humans progressively present metabolic disorders, such as the metabolic syndrome, type 2 diabetes (T2D), non-alcoholic fatty liver disease, certain types of cancer, cardiovascular disease and Alzheimer´s disease, wherever the sedentary lifestyle spreads in the world12–17. For more than 2.5 million years, our metabolic adaptation to seasonal food availability was considered to be an outstanding survival advantage. Nowadays, the same survival strategy in a completely altered surrounding is responsible for a constant body fat accumulation for periods of food deficiency, which will very likely not appear again.\n\nIn this article, we argue that the metabolic epidemic in the developed world is based on a deficit in metabolic flexibility, a term initially stated by Kelley and Mandarino18, which describes the way human physiology is adapted to alternate between lipid and carbohydrate fuels to cope with discontinuities of energy disposability in the environment. Here, we conceive this concept not only in a cellular but also rather systemic intention, and hypothesize that the modern energetic inflexibility, typically displayed by symptoms of neuroglycopenia, may be reversed by recultivation of suppressed metabolic programs, now obsolete in an affluent environment. In a model of predominant flux of energetic substrates between organs, the basic metabolic programs of humans’ primal hunter-gatherer lifestyle are opposed to the current sedentary lifestyle (see Figure 1 and Figure 2). In particular, we suggest that those metabolic programs relying on efficient fatty acid and ketone body oxidation are most of the time shut off in the modern lifestyle and have to be reintegrated in order to overcome the obesity epidemic – widely known as the breeding ground for most of the Western diseases (WD)19–22.\n\nThe brain orchestrates its energy needs by either “pulling” energy from storage organs (from inside the body) in the daily foraging mode, in the short-term fight-flight mode or during long-term hunger in the starvation mode. If food is available, the brain is supplied with energy substrates through a “push” of nutrients (from outside the body) in the rest mode. If all programs are activated occasionally, the metabolic system holds the energetic equilibrium and body weight remains stable.\n\nThe “brain pull” (energy allocation from storage organs to the central nervous system) is compromised by “pushing” energy-rich nutrients into the metabolic system continuously. As a consequence, the foraging and starvation modes are both permanently unattended, which leads to a constant fueling of adipocytes, and in the long run to lipotoxicity, sarcopenia, low-grade inflammation and all associated metabolic diseases.\n\n\nSurvival of the most flexible\n\nEvolution shaped the human metabolic system for ancient times of frequent energy deficiency rather than the present opulence, which entails adverse consequences, such as hyperglycemia, hyperinsulinemia, dyslipidemia and a chronic proinflammatory state induced by, among other things, accumulating visceral body fat21,23–26. Hunger seems an adaptive response to food deprivation that involves neuroendocrine changes to motivate and enable food-seeking behavior27. If evolution would not have developed a basic need of hunger-induced motivation for voluntary exercise in correspondence with energy allocation to the locomotor system, Homo sapiens would have become extinct7,28. When ATP-levels of neurons decrease, the provision of energy to the brain enjoys a privilege, due to limited storing options in cerebral tissues. As a consequence, the metabolism must favor an ongoing energy supply to the brain in any kind of situation29–31, whereas skeletal muscles, important in starvation periods to sustain foraging, can supply themselves with energetic substrates using intramyocellular lipids for ß-oxidation32. Accordingly, if humans’ physical integrity would depend on a continuous ingestion of foods, humankind would not have been able to withstand climate changes, seasonal fluctuations, different vegetation zones, physical conflicts with other hominoid groups, infectious diseases, and intoxications of rotten or dangerous food sources. Along these lines, it has been argued that flexibility of the skeletal muscles to switch from predominantly lipid oxidation (in resting conditions, during fasting or low carbohydrate intake) to increased glucose uptake and storage under insulin-stimulated conditions, was one of humans’ major survival advantages18,33,34.\n\nStoring excessive calories as fat in affluent times is an existential meaningful adaptation in the context of food shortages. Based on this hypothesis, which has been discussed controversial in the field of anthropology, the geneticist James Neel postulated in 1962 his much-cited thrifty gene hypothesis, which states that genes have been positively selected for efficient intake and utilization of food − good for hunter-gatherers in a feast/famine environment, bad for modern people in a world of plenty. In his field studies on Yanomami Indians of the Amazon region, Neel observed that individuals were completely free of overweight and T2D35–37. In contrast, the incidence of T2D in Western industrial countries is at an alarming status15,38. Obviously, people currently face a dramatic mismatch between their genetic heritage and today's sedentary lifestyle.\n\nProponents of Neel´s approach consider recurring famines as the driving force for the natural selection of thrifty genes9,39,40. Neel proposed that muscles become insulin resistant in a metabolic starvation program to maintain high glucose levels for the central nervous system, while lipolysis is upregulated to provide fatty acids for muscles and ketone bodies for the brain. Speakman challenged this hypothesis later on41 with the counter-argument that if overweight would have been so beneficial in the past, there wouldn’t exist so many slim people nowadays. He opposed the so-called predation release hypothesis and blamed the loss of natural predators for the obesity epidemic, which was forced back around two million years ago by the development of social behavior, weapons, and fire37,41.\n\nRecently, the hypothesis of Neel gained new evidence when Johnson et al.42 found a mutated gene connected with stockpiling. According to the authors, this mutation did not originate in the Paleolithic age but rather before 13–17 million years ago, when human ancestors were still great apes and living conditions became colder42. Suddenly, fruits were scarce during the winter period. Interestingly, neither humans nor apes hold a functional gene that expresses the enzyme uricase, as in most animals43. Uricase degrades uric acid, which is produced by the breakdown of DNA substances from food or body's own cells44. Therefore, humans display higher uric acid levels than animals, but obese individuals show much higher levels of uric acid as slim fit people45. Watanabe et al.46 demonstrated that blood pressure increases when uricase is inhibited by medication. Furthermore, a persistent uric acid overload provokes inflammation in the kidneys, which blocks sodium excretion. This mechanism is responsible for the development of hypertension.\n\nIn the animal kingdom, those animals most qualified for fat accumulation share the best chances of survival. Aside from increasing uric acid levels, Ishimoto and Lanaspa47 discovered that if rodents were administered a fructose-rich diet, they would eat more and move less due to disturbed satiety signaling. A medication-induced reduction of uric acid not only lowered blood pressure, but also decreased typical signs of the metabolic syndrome (high blood sugar, high triglycerides, high blood pressure and low HDL-cholesterol). In addition, the same group also showed in vitro that a medication-induced uric acid level reduction impedes liver cells to convert fructose into fat compounds. As winter periods became dry and cool in the middle Miocene (approximately 15 million years ago), Hominoids became able to metabolize fructose into fat. However, a survival advantage in the past has now become a curse. Since the invention of the enzymatic extraction of fructose from corn in the 1980s, refined fructose is attached to most industrial food products, especially sugar-sweetened beverages48,49. Today, lipogenesis-promoting fructose-glucose syrup is hidden in most industrial food substances. As in Paleolithic times, no winter or dry period provides a degreasing any longer. Apparently, the human metabolic equilibrium seems to be literally depending on differences in food availability, which implies an alternating depletion and reload of energy storage. Anyhow, the uricase mutation occurs as strong evidence for the renaissance of Neel´s thrifty gene hypothesis.\n\nRegardless of the genetic debate, obesity establishes where industrialization arrives50. In this context, a clear trend can be observed in developing countries. If people leave their rural habitat and move to the cities, obesity and T2D are on the fast lane13,17,51. Modern inventions to make people’s physical life more convenient are extremely successful (pedelecs, ready-to-eat-food, home automation etc.). When modern humans obtain the opportunity to save energy, they unconsciously select convenience, e.g. parking close to the entrance of a shopping center to avoid walking distances, favoring packaged food instead of spending hours preparing meals. Undoubtedly, this energy-saving behavior must have been shaped by evolution based on fluctuations in energy availability. The screw of time cannot be turned back, but the crucial question is: How can we remain metabolically healthy in a digital and saturated environment, which promotes sedentary behavior?\n\nAnthropologic evidence suggests that, in the course of humanization, humans’ brain size increased at the expense of a shorter gut52,53. Presumably, humans found more energetically efficient and brain building nutrients54,55. In this regard, a long intestine became obsolete with the advantage to expend less energy on digestion, which allows energy distribution to a growing, energetically more demanding brain and the immune system53,56,57. It is not by coincidence that glucose is the first macronutrient absorbed in the small intestine. Although hypothetical, this fact could underline the rarity of sugar in ancient times. At any rate, cerebral neurons usually favor glucose as their primary energy substrate. However, they are capable of either utilizing ketone bodies from visceral fat cells in starvation periods56,58,59 or muscle cell-derived lactate in intense activity (see below). Ketone bodies are the major alternative fuel source for the brain, providing approximately one third of its energy requirements after only four days of very low carbohydrate intake60. Ketone utilization is thereby increased in proportion to ketone body blood concentrations and inversely related to the brain’s glucose utilization. One mechanism seems to involve a downregulation of glycolysis in astrocytes, which sit between neurons and the vasculature, in this way securing a steady glucose supply to neurons61. Interestingly, astroglia and neurons seem to be capable of producing ketone bodies by themselves under conditions of increased AMPK activation, such as hypoxia or hypoglycemia62.\n\nIn this context, the selfish brain clinical research group (Lübeck, Germany) differentiates between ingestive and allocative behavior (see Figure 1 and Figure 2). In other words, if a human being’s brain is adapted to satisfy its energy demands by ingesting high-glycemic dense nutrients (push-principle), it forfeits the ability to allocate (pull-principle) substrates from peripheral storage organs, i.e. glucose and ketone bodies from the liver, free fatty acids (FFA) from adipocytes and lactate from skeletal muscles31,56,58,63,64. Instead, in affluent, sedentary conditions high-glycemic load foods are continuously pushed into the metabolic system (see Figure 2), promoting ongoing ingestive behavior, followed by insulin and leptin resistance in the long run. Constant food availability and physical rest accompanied by chronic psychosocial stress insidiously impairs allocative behavior, leading to overnutrition, caloric excess and a persistent fueling of adipocytes. As described, such metabolic inflexible individuals often perceive hypoglycemia (neuroglycopenia), answered by further food intake – the beginning of a vicious circle.\n\n\nThe hunter and gatherer lifestyle\n\nForaging is the vital need to search for food based on locomotion, which requires energy expenditure in the form of running, sneaking or sprinting to plunder big or small game or climbing and stooping in order to collect honey, berries, nuts, seeds and plants. Admittedly, human beings’ ancestors always had to balance their investment of stored energy against the assumed outcome of a supposed foraging success65 to get physically active. Independent from hunger, thirst or other motivational drives, the circadian rhythm ensures the initiation of the so-called cortisol awakening response66. One major task of the glucocorticoid cortisol is binding to fat cells and glycogen storing tissues in the liver and skeletal muscles, when blood glucose concentration is low67. Cortisol levels increase autonomously in the morning before awakening to provide preferentially glucose for the brain and fatty acids for skeletal muscles to support foraging behavior, mainly based on fat-consuming type 1 muscle fiber activity. Glucose represents the energy substrate for type 2 muscle fibers, necessary for survival in lethal fight-flight-situations29,68,69. While this can be considered a seldom event in Paleolithic times, today´s long-lasting psychosocial stress in an economically growth-driven sedentary environment leads to prolonged cortisol release, stimulating gluconeogenesis but without subsequent glucose clearance by skeletal muscles70.\n\nThe light-dark cycle forced organisms to develop a circadian rhythm to save energy and avoid predation by specialization to an active (light) and inactive (dark) period71. Physical activity is increased in the light phase and, apart from foraging, also includes behaviors such as hazard avoidance, nest building, fight-flight situations, etc. All behavior patterns in the context of physical activity are associated with energy demands of neurons and skeletal muscles, although energy intake was never predictable before the era of agriculture. As a result, to maintain homeostatic levels in terms of different environmental stimuli, humans inevitably became highly flexible in energy allocation72. In the morning, the circulating amount of glucose rises, a fact known as the dawn-phenomenon73. Simultaneously, the non-insulin-dependent glucose utilization is increased in skeletal muscles. Even muscles at rest demand more glucose in the morning than in the afternoon74. Animal studies show that if the metabolic rhythm is disturbed, e.g. due to night shifts or by exchanging the physical activity phase with the rest phase, the risk of glucose intolerance and obesity increases75. In contrast, fasting during the activity phase prevents metabolic disorders76. Those insights illustrate that autonomic endocrine functions prepare human organisms in the morning for physical activity independent of energy intake.\n\nAcross cultures and epochs, people always tend to consume three meals a day77. At daytime, leptin levels decrease and hunger signals emerge to increase sensitivity to brain reward systems, which boost the motivational drive of physical activity, facilitating foraging behavior78–80. Furthermore, lipolysis is upregulated due to activation of the hypothalamic-pituitary-adrenal (HPA) axis, the central nervous system and glucagon from pancreatic islets to induce ß-oxidation of fatty acids, breakdown of glycogen and gluconeogenesis. In contrast, during night time, leptin levels increase, since appetite needs to be reduced in order not to disturb sleep by hunger signals. In addition, due to the fact that the activity of the HPA axis and the suprachiasmatic nucleus (SNS) are low, ketone body utilization provides energy for the brain, while growth hormone stimulated gluconeogenesis allocates glucose to the immune system81–84.\n\nResponsible for controlling the circadian rhythm is the SNS. This core area of the hypothalamus can activate or silence various organs via the central nervous system. Both, the sympathetic and parasympathetic part of the central nervous system can subtly distinguish between intra-abdominal or subcutaneous fat by activating storage types depending on the vital necessity of the body71,77. The vastest release of adiponectin, produced by adipocytes as a fundamental signal for the brain to modulate food intake85, occurs at 10 a.m. in the morning, synonymous with the greatest glucose tolerance86. Taken together, in the morning, human organisms are metabolically well prepared for physical activity, independent of energy intake. Accordingly, it seems contradicting that leading authorities throughout the developed world recommend high-glycemic foods for breakfast in order to provide energy for a sedentary daily routine, since cortisol has been generating energy at sunrise for more than 2.5 million years without energy intake66,87,88.\n\nAs all living organisms, humans maintain a dynamic homeostasis, which is constantly challenged by internal and external stressors89. The fight-flight response represents the archaic physiologic response to a threat from a danger signal90,91. In the history of mankind, humans always faced external threats, such as predators, storms, fire, hostile peers, and toxins in plants, meat or water, as well as internal immunological hazards, such as viruses, bacteria or fungi92. Dangerous situations were usually solved within minutes/hours/days (e.g. escape from predators) or at least weeks, because fighting acute infections or healing trauma could only last until all energy stores were empty, which means approximately 19 to 43 days for females and 28 to 41 days for males6,93.\n\nIn general, fighting and fleeing require augmented energetic demand to support locomotor behavior. Energy allocation is a prioritized issue in this context. If rapid muscle activation is important for survival, the SNS allocates energy to the brain, heart and large skeletal muscles91,94. Blood flow in muscles can increase from 1200 to 22,000 ml/min. In contrast, blood flow to organs such as the kidneys or the viscera, which are energetically neglected in acute danger situations, decreases significantly69. The interplay of SNS-derived catecholamines and the secretion of cortisol, delivered by the HPA-axis, mobilizes glycogen and fatty acids from storage organs (muscles, liver, fat tissue) to provide fuel for the locomotor system. The reversion of an upregulated stress system to homeostasis is metabolically demanding. In case of an acute infection, each degree above normal body temperature costs a daily metabolic increase of 7–13%68,95,96. Already moderate infections boost gluconeogenesis up to 150–200% and severe infections cost 15–30% of body weight due to a persistent elevated basal metabolic rate of 25–55%97,98. Consequently, based on volatile environmental conditions, it becomes clear that humans’ Paleolithic ancestors could not afford a chronically activated immune system, because hypermetabolism during an acute inflammation would become life-threatening if energy stores run empty. However, modern danger signals (e.g. psycho-emotional stress, high meal frequency, physical inactivity) chronically affect humans’ immune system99–101. This persistent low-grade inflammation state is displayed by the mounting epidemic of non-communicable diseases in Western societies102–104.\n\nIn acute danger situations, type 2 muscle fibers demand high amounts of glucose to initiate fighting or fleeing behavior18. In order to avoid a glucose conflict between the brain and contracting type 2 muscle fibers, evolution created alternative pathways to secure the energy demand of the brain. During high-intensity work, type 2 muscle fiber derived lactate is increasingly adopted and oxidized by type 1 muscle fibers and organs such as the heart and brain105 or – to a lesser extent of up to 20% clearance – recycled to glucose in the hepatic Cori cycle106,107. Similar to ketone bodies, brain lactate uptake is accomplished via monocarboxylate transporters, proportional to the arterial concentration of lactate, and able to reach a similar rate as glucose uptake, yielding up to one third of the carbon sources for oxidative phosphorylation108. Frequent exercise improves lactate removal106 which subsequently may stimulate the production of brain-derived neurotrophic factor and improve cognitive performance109.\n\nThe flexibility to switch between metabolic pathways within seconds, prevented metabolic impairments of people’s physical and cognitive performance, which was vital for survival in a hostile environment5,33. Danger and stress factors, affecting the Paleolithic ancestors, were acute within a relatively short timeframe and induced physical activity (escape) or sickness behavior and anorexia in case of an acute inflammation to overcome major wounds or severe infections as soon as possible110. Certainly, nowadays, modern humans suffer from chronic mental stress, which can last for days, weeks or even years with severe metabolic and psychiatric consequences92,111,112.\n\nUnlike today, human beings’ Paleolithic ancestors could not predict meals. Human nutrition used to be subject to weekly, monthly as well as seasonal fluctuations113–115. Anyhow, in periods of fasting, humans should have been able to use stored energy, if necessary, for many hours or even days without any food supply. In other words, if pushing exogenous energy into the body used to be impossible due to food shortage in the past, energy allocation from energy stores to target tissue seems the only alternative to survive famines. For that reason, in late summer or at the end of a rainy season (the time of plenty), it became extremely important for our Paleolithic ancestors to take advantage of easy digestible carbohydrates, such as ripe fruits, to build up body fat, preparing for a scarcer food supply during winter or dry season (the time of caloric or carbohydrate restriction)116. Homo sapiens adapted to these seasonal changes by developing several survival mechanisms. One example is given by the fructose transporter, which enables fruit-derived fructose to be allocated into cells independent of insulin117. In contrast to glucose (20%), nearly 100% of all ingested fructose is immediately converted into fatty acids and then further esterified to storage triacylglycerols by the liver45,118–120. Adipose tissue, responsible for 60% of all triacylglycerol accumulation, can store large amounts of excess FFA. The mechanism to convert fructose into fat was an important survival advantage for our ancestors, particularly if facing seasonal starvation periods. In the animal kingdom, hibernating species were not the only species that developed mechanisms to store energy, and protect themselves in times of food shortage. For example, northern elephant seals can fast for 60 days121 and king penguins survive 5 months without energy intake27. Adipose tissue of modern humans contains approximately 13 kg fat or 500,000 kJ, which would theoretically last for 2.4 months57. As a result, survival was not only dependent on fitness, as argued by Charles Darwin122, but also on fatness, since overweight can guarantee weeks of survival without any food supply123. Alternatively, it can be imagined that early humans had to endure times not necessarily of reduced energy intake, but drastically reduced carbohydrate intake, if animal, but not plant foods were abundant. Physiologically, this situation is similar to fasting because it is mainly (exogenous) glucose that interferes with the adaptive response to fasting, while fat is a neutral macronutrient in this respect124. The nuclear peroxisome proliferator activating receptors that stimulate fat oxidation and ketogenesis are activated by FFA, which can stem from both exogenous, preferably obtained from hunted animals, or endogenous fat, released during times of food scarcity125. It is interesting that across cultures, indigenous humans share a high preference for animal fat126,127, underscoring the importance of this macronutrient for human health and survival in times when only animal food was available.\n\nThus, intermittent fasting and longer-term caloric as well as carbohydrate restriction are parts of our genetic heritage. All three can lower glucose, insulin, triglycerides and lipid accumulation and, at the same time, increase insulin sensitivity, while increasing HDL-cholesterol128. Intermittent fasting taken by itself was proven to reduce low-grade-inflammation significantly by degrading IL-6, TNF-alpha and CRP114,129,130. Studies with mice also demonstrated that time-restricted feeding without reducing caloric intake can prevent metabolic diseases, if fed a high-fat diet76. Other evidence reveals that intermittent fasting and caloric restriction both enhance cardiovascular function and increase insulin sensitivity123,131. In humans, glucose homoeostasis in fasting periods are initially bypassed by liver glycogen (0.5 days) and proteolysis mainly from muscles (from day 1 to 3). From day 3 onwards, liver-derived ketone bodies are used as a glucose substitute for the brain, muscles and immune cells82. Recapitulating, fatness is a fundamental survival strategy against hunger, which all creatures developed during biogenesis to survive starvation periods. Nature provides seasonal fluctuations of food availability, leading to alternating periods of fatness and leanness, whereas today, energy abundance determines modern life throughout the year.\n\nAs mentioned before, food seeking behavior is an evolutionary-based motivational drive to forage, in order to maintain energy balance on homeostatic level. Foraging success in the form of food represents the primal reward for a physical effort, which activates parasympathetic circuits to promote digestion, recovery of energy stores and repair110,132–134. In order to avoid overnutrition, our metabolic homeostatic system involves several hormonal regulators of hunger and satiety, such as leptin, ghrelin, and insulin, which act on hypothalamic and brainstem circuits to inhibit further feeding. Dysfunction of this homeostatic system, such as leptin resistance, can result in a persistent state of positive energy balance, overeating and obesity. Palatable high caloric foods might be a major reason for this dysfunction in the Western world by exchanging the regulation of food intake from homeostatic to rewarding pathways111, since food intake displays much more than balancing energy status. Humans consume food also for its hedonic properties independent of energy status, revealing that the brain reward system plays an important role in feeding behavior135. A pivotal component of reward and motivation circuitries are cerebral neurons, which originate in the ventral tegmental area. These cells send projections to the nucleus accumbens, a structure deep beneath the frontal cortex, by utilizing the essential neurotransmitter dopamine. Termed as the mesolimbic dopamine system, this brain region is responsible for the processing of aversion, motivation, pleasure and reward, and in consequence is linked to encoding rewarding stimuli, such as comfort food, sex, sports (runner´s high), and addictive substances (caffeine, ethanol, nicotine, heroin, etc.)132,134,136. One hypothesis for abnormal hedonic behavior displayed by overconsumption of high-glycemic carbohydrates could be that modern processed foods lack in essential amino acids. In addition, processed foods contain less micronutrients compared to the Paleolithic food selection137,138. A poor nourishment status in both essential macro- and micronutrients might be at least co-responsible for reward deficiencies, which result in ongoing food seeking behavior rather than resting after enjoying the foraging success.\n\nEnjoying palatable food is a powerful motivational drive that can override homeostatic signals. Affluence of energy-dense food is considered a major environmental risk factor for obesity139. Rats with extended access to palatable food rapidly gain weight and reward deficits132. Leptin resistance could be the functional link between reward deficiency and persistent ingestive behavior. Leptin receptors are expressed on dopamine neurons in the ventral tegmental area140. As proven in rodents, leptin infusions into the ventral tegmental area inhibit the activity of dopamine neurons and decrease food intake141. Further, knockdown of leptin receptors in the ventral tegmental area enhance food intake and preference for palatable food141. Administered from adipocytes, leptin acts as an inhibitor of mesolimbic dopamine transmission in physiological conditions to interrupt food seeking behavior if storage organs are loaded. Obese individuals demonstrate increased activation of reward circuitries in response to palatable food compared with lean controls142,143. Apparently, hypersensitivity of reward circuitries predisposes to overnutrition and weight gain144. However, during the further weight gain process, rewarding effects are blunted to reach hyposensitivity, which perpetuates overnutrition in order to overcome reward deficits145,146. In conclusion, effort and reward are directly interconnected to silence dopamine-driven behavior. In Paleolithic times, reward substances were limited, whereas today modern humans are surrounded by a plethora of rewarding stimuli in a nearby environment, such as refined sugar, alcohol and plenty of substances, which can be addictive and finally inhibit physical activity.\n\n\nThe sedentary lifestyle\n\nThe current metabolic dysplasia is descriptively explainable with a neglect of the important ancient metabolic programs. Our metabolic flexibility qualified us to be the most dominant of all living species on Earth. Excluding agricultural revolution, humans’ energetic flexibility fell victim to major technical achievements of the last 150 years, a blink of an eye in an evolutionary point of view. For clarification, since 1887 food is stored in refrigerators, since 1900 muscle-derived energy expenditure is saved by the use of cars, and since 2001 people meet friends all around the world via the world-wide-web without investing a single calorie in locomotion. The inventions of the last three centuries increased physical inactivity. Moreover, the launch of high fructose corn syrup (HFCS)-production in the late 1980s has set up the crown on a sedentary lifestyle with abundance of nearby and easy to digest energy. Ever since, the obesity epidemic proceeds relentless4,119,147,148.\n\nApart from energy-dense food availability and ongoing mechanization, the digital revolution has led to another important underlying factor concerning obesity. This factor is strongly related to how fast the professional life has turned to be. Many modern humans capitulate from the speed of innovation. Stress-related disorders are increasingly exorbitant149,150. Chronic psycho-emotional stress factors, such as liabilities, job loss or time pressure, face an archaic stress system (fight-flight-mode), made to overcome acute danger situations rather than long-term stress. Locomotion, the evolutionary designed solution to dismantle acute stress reactions, is no longer an option in a sedentary working ambiance. As a result, sedentary lifestyle in association with chronic psycho-emotional stress and a plethora of high-glycemic load foods, lead to a constant fueling of adipocytes and tissues (see Figure 2), which can store fat in areas such as the liver, heart, skeletal muscles and the vascular system151. By contrast, skeletal muscles, which are able to increase their basal metabolic rate after physical activity57,152, atrophy with age in modern societies. Life-threatening diseases, such as cancer and dementia, are characteristic examples, where sarcopenia is a cardinal symptom. Accordingly, we blame here three essential lifestyle factors for obesity and its related diseases:\n\n1) chronic high-glycemic food availability in the nearby environment\n\n2) loss of existential motivation for physical activity\n\n3) increase of psycho-emotional stress not ventilated by muscle activity\n\nThe modern lifestyle promotes continuous fueling of adipocytes (see Figure 2). In response to adipose hypertrophy, neutrophils, macrophages, natural killer cells and other immune cells infiltrate adipose tissue and in the further process develop an inflammatory environment, based on a phenotypic switch from anti-inflammatory M2- to pro-inflammatory M1-macrophages86,153. This condition induces a systemic low-grade inflammation, characterized by high levels of TNF-alpha and IL-6, which contributes and amplifies insulin resistance19,21,23,154,155. As accumulation of adipose tissue approaches its limit, the body is compelled to use organs and other tissues for fat storage, such as skeletal muscles, pancreas, and liver, and as a second choice the heart, kidneys and bones, thereby boosting insulin resistance23,156–158. As a matter of fact, the liver is one of the organs affected first. A non-alcoholic fatty liver (NAFL) is present in nearly 25–50% of the general population in Western countries158,159 and strongly related to T2D, hyperglycemia, hyperinsulinemia, low HDL-cholesterol and represents an independent risk factor for cardiovascular diseases160. On a related note, changes in lifestyle patterns have been more successful in the context of a NAFL than pharmacological treatments, whereas exercise in isolation has not been proven to be effective161.\n\nGlucose is the primary nutrient for neurons and red blood cells, but rather rare in our primal habitat. Therefore, honey has always been a privileged food for hunter-gatherers, and accounts for a substantial proportion of kilocalories in some primal living societies, since honey is the most energy dense food in nature. In the Hadza diet (a hunter and gatherer tribe in Tanzania), honey contributes roughly 15% of their total energy intake162. At least in warm areas, honey might have been a regular part of humans’ hunter-gatherer past, since current indigenous people take not inconsiderable risks to gain access. All of humans closest relatives, like chimpanzees, bonobos, gorillas and orangutans, consume honey. Honey is the sugar source par excellence, as its dry matter contains 95% of carbohydrates, averaged 40% fructose and 30% glucose163. For this reason, honey is the ideal fuel to supply the central nervous system with glucose and simultaneously refill fat stores with fructose for both animals and humans, living in the wild to stand seasonal fluctuations of energy availability. Today, fructose is ubiquitous as a low-cost sweetener. In the late 1980s, the US food industry began incorporating HFCS into soft drinks and industrial foods, greatly promoting the persistent caloric surplus in present day societies164. It has now become clear that regular consumption of processed and high-glycemic load foods, especially sugar-sweetened soft drinks45,48,165, supported by a growing lack of exercise, change human body composition towards an accumulation of visceral fat mass and sarcopenia34,166. As a result, the reduction of sugar-sweetened foods might be one of the major key factors to overcome the obesity epidemic.\n\nThe capability to store fat inside muscles could have been an evolutionary advantage to preserve the existential motivation to hunt, fish and gather food in times of shortage. Observational studies of recent hunter-gatherer societies, such as the Hadza in Tanzania, forager groups of the Andaman Islands or the San people in the Kalahari Desert, reveal that men usually walk long distances for hunting animals, while women search for collectable food and fresh water in the nearby environment65,167–169. Most physical activities of the mentioned aboriginal tribes are movements at low intensities, where skeletal muscles use fatty acids as the primary energy source. Oxidative type 1 muscle fibers are characterized by a higher fat content than glycolytic type 2 fibers. They contain more mitochondria, which utilize stored intramuscular fat as a rapidly available energy source. Therefore, oxidative slow-twitch muscle fibers are perfectly designed to overcome long distances in order to forage or see new biospheres. Interestingly, high intramuscular fat is associated with both the development of insulin resistance and the effects of endurance training, known as the training paradox in sports physiology170. Although fat accumulation in muscles of obese people is a consequence of a spillover of nutrient excess in sedentary terms, it has been suggested that the supercompensation of intramuscular fat after fasting periods or exhaustive workouts could have been beneficial in human evolution to permit physical activity in times of food shortage32. De facto, if prolonged exercise (hunting big game or discovering new hunting grounds), comparable with present-day sporting challenges, such as marathons or ironman triathlons, was vital for survival in the past, high intramuscular fat stores secure ß-oxidation in active muscle cells18,33. In this situation, glucose can be spared for the central nervous system (at daytime), glucose-depending cells (e.g. red blood and kidney cells) and the immune system (at night) – an excellent metabolic division of work on the scarcity of glucose to guarantee a constant glucose influx to glucose-depending cells57,171,172.\n\nNowadays, the biggest metabolic challenge for obese, (pre-)diabetic, metabolically inflexible healthy individuals, but also athletes on high carbohydrate diets, is to maintain blood sugar levels in an affluent environment. In conditions of high locomotor speed, chronic mental activation or insulin resistance, there is an increasing demand of glucose either by type 2 muscle fibers (in high-intensity conditions) or the brain (in chronic mental activation or under insulin resistance). In dangerous situations, when muscle activity beyond 70% VO2max is needed for survival (or nowadays in competitive sports), the utilization rate of muscle and liver glycogen can come to its limits33,56,171. Consequently, the euglycemic state is compromised and metabolically inflexible athletes typically show symptoms of neuroglycopenia, which are commonly answered or prevented by the intake of carbohydrate-rich foods. This situation would be different in “keto-adapted” athletes, who are able to sustain fat oxidation at high intensities and display high levels of ketone bodies during exercise, which can be used by the brain. These capabilities have been impressively documented by Volek et al.172, showing that in keto-adapted endurance athletes peak fat oxidation is shifted towards higher intensities (70 ± 6% of VO2max) and displays absolute values more than twice as large (1.2 ± 0.2 g/min) as those of endurance athletes on a high carbohydrate diet. Apart from athletes, owing to the digital sedentary lifestyle that affects all aspects of life, modern humans frequently walk less than 1 km per day, their physical activity level has fallen below 1.7, and their VO2max fitness markers are of alarming status1–3,10,173,174. In such conditions of physical inactivity, the need for carbohydrates as a fuel is at a minimum level. By contrast, most authorities in the Western world recommend at least 50% of the daily caloric intake as carbohydrates for a sedentary lifestyle175–178. Although neurons demand 20% of the total daily glucose turnover, the brain is not capable of accumulating large amounts of glucose. Since high blood sugar levels are pro-inflammatory9,179,180, excess glucose, if not utilized by the brain or muscles, has to be metabolized in the liver as fat, continuously fueling adipocytes (see Figure 2)\n\nEven humans with a constitutional high percentage of fast-twitch fibers, which are competent to burn high amounts of glucose within a short timeframe, have given up this opportunity to clear glucose from the blood in a digital world. These subjects rapidly store fat given a Western diet in physical rest33,181–184. In Paleolithic times, this adaptation could have been a survival advantage in specific environmental conditions, but, nowadays, it seriously impairs metabolic health.\n\nLike all biological systems, physical and mental stress are mutually dependent on the dynamic homeostasis. For example, it is well established that an overload of exercise activates humans’ immune system and provides a higher vulnerability to infectious diseases185–188. In modern societies, physical workload is increasingly detached from intellectual workplaces. In the light of working conditions at present, a significant trend can be observed that once humans enter sedentary professions, they become overweight189. Nowadays, in an affluent environment under the influence of multiple new and unratable stressors, people’s primal systems can come to their limits. Social hierarchy is a perfect example to display individual stress load. Sapolsky190,191 found that in baboons social subordinance and social isolation is associated with manifestations of hypercortisolism. In modern working organizations, the mean cortisol level is higher in individuals with low socioeconomic status because of high job demands. The same holds true for low job control in general192. Many overstressed people experience long-term stress since they are not capable of changing their self-chosen or enforced environment193. From a biological perspective, intense muscle activity is the natural response to stressful or dangerous stimuli. At the present day, exercise has lost its primal role due to the fact that modern humans living in a digital environment do not secure their subsistence by physical workload anymore. Exercise no longer serves as either foraging behavior or escaping from dangerous conditions and fighting predators. If modern humans are mentally hyperactive (e.g. chess playing on world class level), skeletal muscles become insulin resistant for a short time, to redirect glucose towards the central nervous system194. In this case, humans’ brains rapidly demand glucose replenishment when stressors continue31,95,195. In the long run, a temporary functional insulin resistance, which secures survival of neurons in acute danger or infectious situations, passes over into a persistent insulin resistant state. This condition was recently viewed as protective to avoid abundant intracellular substrate accumulation, especially in liver and muscle cells196. However, as a consequence of prolonged insulin resistance, insulin receptors are blunted and transmembrane signaling is attenuated197–199.\n\n\nConclusions\n\nA return to Stone Age conditions is neither possible nor desirable. Plants are highly cultured, movement has lost its existential necessity to forage, and stress influences act no longer as acute danger signals but are subtly persistent. Paleolithic times were certainly no paradise. Survival always hanged by a threat. Modern achievements such as food preservation, mechanization, antibiotics, high-tech medicine and the virtual elimination of infant mortality have significantly increased life expectancy in the Western biosphere200. Even contemporary hunter-gatherers do not reach the age of modern humans, predominantly due to much higher rates of infant mortality and serious, unhandled infections201. Nevertheless, the era in which modern humans suffer from chronic diseases is extending202 and metabolic disorders occur much earlier16,21,203,204. Periodic fat gain is considered physiologic, whereas persistent overweight contradicts humans’ evolutionary designed metabolic flexibility and promotes a chronic low-grade inflammation, which can be considered as the hotbed for WD. Apparently, since evolution is a slow-acting process, modern humans are not yet well adapted to a sedentary lifestyle. Approximately 99.5% of our existence, we lived as hunter-gatherers1,2, which means being metabolically prepared for any kind of environmental condition. According to the present article, today´s sedentary lifestyle limits our primal metabolic flexibility to a stress and rest mode (see Figure 1 and Figure 2). If not consciously integrated in daily life in the form of periodic fasting, fasting mimicking diets, such as a ketogenic diet, or exercise programs, the starvation and foraging mode are consistently neglected. To maintain metabolic flexibility in a modern habitat, characterized by energy abundance, prolonged psychosocial stress and physical inactivity, people should retrain all described modes periodically, which secured our survival in the wild. We argue that no specific diet, exercise or anti-stress program must be followed if behavior is adjusted based on human being’s evolutionary heritage.", "appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nO'Keefe JH, Vogel R, Lavie CJ, et al.: Achieving hunter-gatherer fitness in the 21(st) century: back to the future. Am J Med. 2010; 123(12): 1082–6. 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PubMed Abstract \| Publisher Full Text\n\nCouto M, Silva D, Delgado L, et al.: Exercise and airway injury in athletes. Acta Med Port. 2013; 26(1): 56–60. PubMed Abstract\n\nWalsh NP, Oliver SJ: Exercise, immune function and respiratory infection: An update on the influence of training and environmental stress. Immunol Cell Biol. 2016; 94(2): 132–9. PubMed Abstract \| Publisher Full Text\n\nHauner H, Bramlage P, Lösch C, et al.: Overweight, obesity and high waist circumference: regional differences in prevalence in primary medical care. Dtsch Arztebl Int. 2008; 105(48): 827–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSapolsky RM, Alberts SC, Altmann J: Hypercortisolism associated with social subordinance or social isolation among wild baboons. Arch Gen Psychiatry. 1997; 54(12): 1137–43. PubMed Abstract \| Publisher Full Text\n\nSapolsky RM: The influence of social hierarchy on primate health. Science. 2005; 308(5722): 648–52. 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PubMed Abstract \| Free Full Text" }	[ { "id": "27936", "date": "27 Nov 2017", "name": "Umesh Yadav", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled “The sedentary (r)evolution: Have we lost our metabolic flexibility?” by Freese J. et al., has discussed an important issue regarding the molecular and evolutionary reasons behind increased incidence of modern life style diseases in human beings. The authors have cited interesting studies to corroborate their points and delivered a convincing molecular and biochemical outlook for Western diseases (WD). The article makes an interesting read for the commoners as well as the scientific community.\nAs far as the deficit in the metabolic flexibility in the modern times, as opposed to prehistoric times, is concerned the arguments given sound rational and justifiable, especially the switch between the fatty acid oxidation verses glucose oxidation in the hunter and gatherers of early times, which the authors argue has been lost in the modern times due to 1., availability of high glycemic food; 2., lack of extensive physical activities; 3., chronic psychological stress.\nThe authors have discussed the relationship between the organs and shift in the metabolic flux during the two different lifestyles, hunter and gatherers, and sedentary. The metabolic linkages with circadian rhythm during hunter and gatherers life style, and how it has got disturbed now, are well explained.\nHowever, there are certain points authors may like to connect to make it more explicable. Take an example where the transition of life style was not abrupt, the hunter and gatherers when became agricultural societies and started dwelling in the stable communities with no uncertainty in most of the life activities, and feast and famine were not as frequent. Did the biochemical changes in the metabolism undergo some adaptation to the relatively and progressively ‘sedentary life style’? The agricultural societies have now transformed into industrialized societies with modern amenities and energy-rich foods within arm’s reach.\nEvolution indeed is a progressive natural mechanism. Is it possible that the ‘thrifty genes’ that became advantageous during hunting and gathering era of human existence, has become a sort of burden and will be eventually eliminated from the gene pool? Probably a better adapted gene set, say ‘lean genes’ to these emerging trends would help humans survive the endemic ‘modern life style’. Are there any indication of such evolutionary tendencies in humans today? Can the modern tools such as CRISPER-Cas be utilized in near future to modify the so called ‘thrifty genes’ and to rewire the metabolic flexibility? This is especially important when the evolution depends more on artificial selection than natural!\nThe life, and the metabolism that drives life, are flexible and would therefore evolve an alternate mechanism to handle the pressure of modern life style on human health and survival. What biochemical alternatives or shunts could be suggested to overcome the so called metabolic inflexibility. Authors may have suggested or postulated more credible ways to thwart the metabolic inflexibility than merely propounding ‘retrain all prescribed modes periodically’, where the term ‘periodically’ is clearly vague!\nAt places the authors have mentioned about the satiety signalling, where they state that leptin resistance is a common scenario in the sedentary life style. Is there evidence that the satiety signaling involving leptin and ghrelin have evolved in favour with the progressing inactive lifestyle?\nThe authors have well explained the brain reward system in feeding behaviour. Modern habits and lifestyle is inclined towards abnormal hedonic behaviour where there is an excessive intake of processed food with high glycemic index and low nutritive value, dissatisfaction to the brain drives such a behaviour leading to obesity. Could authors suggest ways or quote studies where balancing the nutritive value of the food has been attempted such that they regulate the hormonal homeostasis that controls the excessive eating behaviour in individuals?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3384", "date": "02 Feb 2018", "name": "Jens Freese", "role": "Author Response", "response": "We would like to thank all reviewers for their constructive comments and positive feedback to our article. We have addressed their comments as described below, and think that they greatly helped to improve our article further. The authors have discussed the relationship between the organs and shift in the metabolic flux during the two different lifestyles, hunter and gatherers, and sedentary. The metabolic linkages with circadian rhythm during hunter and gatherers life style, and how it has got disturbed now, are well explained. However, there are certain points authors may like to connect to make it more explicable. Take an example where the transition of life style was not abrupt, the hunter and gatherers when became agricultural societies and started dwelling in the stable communities with no uncertainty in most of the life activities, and feast and famine were not as frequent. Did the biochemical changes in the metabolism undergo some adaptation to the relatively and progressively ‘sedentary life style’? The agricultural societies have now transformed into industrialized societies with modern amenities and energy-rich foods within arm’s reach. A: Thank you for this comment. However, our point was not to imply that early agriculture led to better food security and less famine. In fact, early agriculturalists may have experienced more frequent and more severe periods of famine than hunter-gatherers due to the latter’s higher flexibility to change habitats (Berbesque et al. 2014, Biol Lett 10: 20130853 ). This also questions Neel’s thrifty gene hypothesis as we now briefly mention in the revised article. However, the lactase persistence gene and copy number variation in the AMY1 (amylase) gene present examples for rapid biochemical adaptions that, however, only occurred in single genes and do not have a large impact on global physiology. Evolution indeed is a progressive natural mechanism. Is it possible that the ‘thrifty genes’ that became advantageous during hunting and gathering era of human existence, has become a sort of burden and will be eventually eliminated from the gene pool? Probably a better adapted gene set, say ‘lean genes’ to these emerging trends would help humans survive the endemic ‘modern life style’. Are there any indication of such evolutionary tendencies in humans today? Can the modern tools such as CRISPER-Cas be utilized in near future to modify the so called ‘thrifty genes’ and to rewire the metabolic flexibility? This is especially important when the evolution depends more on artificial selection than natural! A: Even if there would be some true aspect to the thrifty gene hypothesis, we think it is very unlikely that evolution would now start to select for “lean genes”, for the following reasons: First, while obesity and its related diseases are now occurring more frequently at very young ages already, they are chronic in nature and do not pose a significant risk for dying before sexual reproduction has occurred. Second, modern medicines and pharmaceutical drugs are able to significantly prolong the life of patients with metabolic diseases. However, whether modern tools such as CRISPER-Cas could be utilized to modify certain “thrifty genes” remains speculative, and we think is beyond the scope of our paper. It would not only presuppose that such genes really exist (our example of uricase is rather unique in its effect on metabolism) but also that modifying these individual genes could reverse or neutralize the effects of an unhealthy lifestyle – which is probably not the case because lifestyle choices affect not individual genes, but have complex effects on the whole genome. The life, and the metabolism that drives life, are flexible and would therefore evolve an alternate mechanism to handle the pressure of modern life style on human health and survival. What biochemical alternatives or shunts could be suggested to overcome the so called metabolic inflexibility. Authors may have suggested or postulated more credible ways to thwart the metabolic inflexibility than merely propounding ‘retrain all prescribed modes periodically’, where the term ‘periodically’ is clearly vague! A: We now mention pharmaceutical drugs such as metformin or “calorie restriction mimetics” such as resveratrol as possibilities that activate the starvation and foraging pathways (AMPK, SIRT1 etc.), but we think it is beyond the scope of this article to provide a somewhat detailed description of these approaches. Also, we are personally convinced that no single drug is able to fully mimic the complex interplay between molecular pathways that gets activated through lifestyle modifications. We have also removed the term “periodically” and replaced it with some more specific recommendation examples. At places the authors have mentioned about the satiety signalling, where they state that leptin resistance is a common scenario in the sedentary life style. Is there evidence that the satiety signaling involving leptin and ghrelin have evolved in favour with the progressing inactive lifestyle? The authors have well explained the brain reward system in feeding behaviour. Modern habits and lifestyle is inclined towards abnormal hedonic behaviour where there is an excessive intake of processed food with high glycemic index and low nutritive value, dissatisfaction to the brain drives such a behaviour leading to obesity. Could authors suggest ways or quote studies where balancing the nutritive value of the food has been attempted such that they regulate the hormonal homeostasis that controls the excessive eating behaviour in individuals?" } ] }, { "id": "28024", "date": "27 Nov 2017", "name": "Johannes Coy", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article ‘The sedentary (r)evolution: Have we lost our metabolic flexibility?’ by Freese and coworkers is an important contribution to solve the huge worldwide problem caused by a dramatic increase of diseases linked to nutrition and life style.\n\nThe authors describe that during the course of evolution, up until the agricultural revolution, environmental fluctuations forced the human species to develop a flexible metabolism in order to adapt its energy needs to various climate, seasonal and vegetation conditions. During the longest time of Homo sapiens’ existence, humans’ daily survival was shaped by food insecurity (feast and famine), adaptation to a widespread range of different food sources (biodiversity), abundant daily exercise frequently under fasting conditions (foraging behavior), as well as unpredictable food supply (intermitted fasting), depending on the daily foraging success.\n\nThe authors underline that the metabolic flexibility safeguarded human survival independent of changes in food availability and food composition. Furthermore the article shows the important and complex interplay and regulation of different metabolic pathways and a very important metabolic switch to the use of ketone bodies and fat oxidation.\n\nIn the course of evolution, environmental stress forced the human species to develop an extraordinary flexible metabolism with multiple programs to guarantee energy equilibrium. Lipolysis, proteolysis, gluconeogenesis, ketone bodies and muscle-derived lactate as alternative energy substrates for cerebral neurons, provide examples by which natural selection tailored humans to buffer fluctuations in energy supply. Hormons as important key regulators of the metabolism as well as the role of the brain are being described in a very good and understandable way. In addition as a result of persistent psycho-emotional stressors the reaction of the brain concomitant with the changes in metabolism is shown. Furthermore the important impact of a lack of exercise is depicted.\n\nDue to this clear description of the metabolic flexibility and the absence of the use of this metabolic flexibility in modern humans following a Western life type, the authors hypothesize that the modern energetic inflexibility, typically displayed by symptoms of neuroglycopenia, can be reversed by re-activation of certain suppressed metabolic programs, which became obsolete in an affluent environment, particularly the ability to easily switch to ketone body and fat oxidation.\n\nThe approach to re-activate metabolic programs represents an important and a not-well known approach to prevent and treat diseases related to metabolism and life style. Therefore this article is of utmost importance for scientists and clinicians in this field, but also enables individual humans to use the knowledge presented in this article to re-activate the metabolic programs by intermitting fasting, changes in food composition and/or exercises e.g. to activate the program to use ketone bodies and fat oxidation.\n\nThere are a few terms which should be considered / could be changed to make the statements more clear.\n\nAbstract:\n\n\"In this article, we argue that the metabolic epidemic is largely based on a deficit in metabolic flexibility.\"\n\nThe term ‘metabolic epidemic’ should be rephrased e.g. In this article, we argue that the strong increase of diseases related with an metabolic impact is largely based on a deficit in metabolic flexibility.\n\nThe same should be done for the sentence in the introduction:\n\n\"In this article, we argue that the metabolic epidemic in the developed world is based on a deficit in metabolic flexibility,…\"\n\nPage 7 \"Adipose tissue, responsible for 60% of all triacylglycerol accumulation, can store large amounts of excess FFA.\"\n\nThe Abbreviation FFA is not being explained\n\nPage 9 \"On a related note, changes in lifestyle patterns have been more successful in the context of a NAFL than pharmacological treatments, whereas exercise in isolation has not been proven to be effective.\"\n\n…the statement ‘whereas exercise in isolation has not been proven to be effective´ should be explained. The meaning of ‘exercise in isolation’ is not clear.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3383", "date": "02 Feb 2018", "name": "Jens Freese", "role": "Author Response", "response": "We would like to thank all reviewers for their constructive comments and positive feedback to our article. We have addressed their comments as described below, and think that they greatly helped to improve our article further. \"In this article, we argue that the metabolic epidemic is largely based on a deficit in metabolic flexibility.\" The term ‘metabolic epidemic’ should be rephrased e.g. In this article, we argue that the strong increase of diseases related with an metabolic impact is largely based on a deficit in metabolic flexibility. The same should be done for the sentence in the introduction: \"In this article, we argue that the metabolic epidemic in the developed world is based on a deficit in metabolic flexibility,…\" A: Done. We rephrased the term as “diseases related to metabolic abnormalities” Page 7 \"Adipose tissue, responsible for 60% of all triacylglycerol accumulation, can store large amounts of excess FFA.\" The Abbreviation FFA is not being explained A: We apologize and have replaced the abbreviation with “free fatty acids” Page 9 \"On a related note, changes in lifestyle patterns have been more successful in the context of a NAFL than pharmacological treatments, whereas exercise in isolation has not been proven to be effective.\" …the statement ‘whereas exercise in isolation has not been proven to be effective´ should be explained. The meaning of ‘exercise in isolation’ is not clear. A: The meaning should be „Exercise on its own without concurrent changes in diet”. However, the whole sentence was removed and replaced by a related sentence." } ] }, { "id": "28692", "date": "06 Dec 2017", "name": "Shuzo Kumagai", "expertise": [ "Reviewer Expertise Health and Exercise Epidemiology", "Exercise Physiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article published by Jens Freese et al. discussed the potential explanations of metabolic epidemic from the viewpoint of the metabolic flexibility sharped by evolutionary through millions of years, and a combination of present-day continuous food abundance, sedentary lifestyle, and psycho-emotional stress. In their manuscript, Jens Freese et al suggested metabolic programs relying on fatty acid and ketone body oxidation are most of the time shut off due to modern lifestyle and have to be reintegrated in order to overcome the obesity epidemic. The storyline of ‘Survival the most flexible’ section could be more clear if the description could be more concise and focusing on how evolution sharped the metabolic flexibility. For example, the content of ‘It’s all about survival: Why evolution shaped metabolic flexibility’ seems discussed the interplay of metabolic programs in brain and other organs (e.g., skeletal muscle and adipocytes) which could be combined into the first section. The sections regarding uricase mutation could also be more straightforward by describing the importance of this mutation (e.g., more efficient metabolism of fructose and fat stores) in the way of evolution.\n\nIn the section of ‘Sedentary lifestyle’, the authors described a modern lifestyle which is opposite to the ‘The hunter and gatherer lifestyle’. Accordingly, this section could also benefit from a more concise storyline by comparisons such as sedentary behavior vs foraging mode. Besides molecular-level evidence, there are also quite a lot of epidemiological evidence has highlighted the importance of environmental, and behavioral (dietary and sedentary behaviors) factors in the progress of metabolic health outcomes. Incorporation of these evidences will provide more insight into how to re-cultivate of suppressed evolutionarily metabolic programs and make the proposed model more convincing and readily understood for the researchers in the field as well as the general readers.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "3382", "date": "02 Feb 2018", "name": "Jens Freese", "role": "Author Response", "response": "We would like to thank all reviewers for their constructive comments and positive feedback to our article. We have addressed their comments as described below, and think that they greatly helped to improve our article further. The storyline of ‘Survival the most flexible’ section could be more clear if the description could be more concise and focusing on how evolution sharped the metabolic flexibility. For example, the content of ‘It’s all about survival: Why evolution shaped metabolic flexibility’ seems discussed the interplay of metabolic programs in brain and other organs (e.g., skeletal muscle and adipocytes) which could be combined into the first section. The sections regarding uricase mutation could also be more straightforward by describing the importance of this mutation (e.g., more efficient metabolism of fructose and fat stores) in the way of evolution. A: We have moved parts from the “Survival of the most flexible” section into the “It’s all about survival: Why evolution shaped metabolic flexibility’” section, and also joined both sections together to better connect the lines of reasoning provided in them. We have also re-structured and shortened the section on the uricase mutation in order to make our argumentation more comprehensible. In the section of ‘Sedentary lifestyle’, the authors described a modern lifestyle which is opposite to the ‘The hunter and gatherer lifestyle’. Accordingly, this section could also benefit from a more concise storyline by comparisons such as sedentary behavior vs foraging mode. Besides molecular-level evidence, there are also quite a lot of epidemiological evidence has highlighted the importance of environmental, and behavioral (dietary and sedentary behaviors) factors in the progress of metabolic health outcomes. Incorporation of these evidences will provide more insight into how to re-cultivate of suppressed evolutionarily metabolic programs and make the proposed model more convincing and readily understood for the researchers in the field as well as the general readers. A: We have removed some redundant text from the first subsection “Modern habitat: A life in mental stress and physical rest” in order to make the storyline and transition to the following subsections more concise. We have also added a total of five new references that provide additional confirmation of our hypothesis that reverting the sedentary lifestyle induces holistic beneficial metabolic effects." } ] } ]	1	https://f1000research.com/articles/6-1787
https://f1000research.com/articles/7-137/v1	01 Feb 18	{ "type": "Research Article", "title": "Nutritional status, survival and mortality in Alzheimer patients - a cross-sectional study", "authors": [ "Elizama de Gregorio", "Dayanna Hartmann Cambruzzi Mendes", "Luan Henrique Patrzyk", "Luana Felski", "Guilherme Barroso Langoni de Freita", "Anne Karine Bosetto", "Bárbara Luisa Fermino", "Maria Vaitsa Loch Haskel", "Flávia Ivanski", "Juliana Sartori Bonini", "Camila Diedrich", "Weber Cláudio Francisco Nunes da Silva", "Elizama de Gregorio", "Dayanna Hartmann Cambruzzi Mendes", "Luan Henrique Patrzyk", "Luana Felski", "Guilherme Barroso Langoni de Freita", "Anne Karine Bosetto", "Bárbara Luisa Fermino", "Maria Vaitsa Loch Haskel", "Flávia Ivanski", "Camila Diedrich", "Weber Cláudio Francisco Nunes da Silva" ], "abstract": "Introduction: Dementia is a common health problem in elderly people, Alzheimer disease (AD) being the most prevalent. AD can be considered as a cause of death and must be registered on the death certificate of the patients. However, most of the time, the main cause of death registered is not related to AD, but as an underlying or contributing cause. For example, individuals who have AD and die from myocardium infarction. This study aimed to analyze if nutritional status was associated with survival and mortality for AD, and if AD was reported as actual cause of death on the death certificate Methods: The study was carried out as a cross-sectional study with elderly citizens of the community registered in the National Health System (SUS), with cognitive, nutritional, biochemical and hematological evaluations of 30 AD patients in Guarapuava, Paraná state, Brazil. Results: Significant differences were not observed between live and dead patients when evaluated considering the methods applied. Only 22% of the death certificates stated death due to AD. The patient’s cause of death showed a strong relation to respiratory issues; potential explanations based on immunological, biochemical and comorbidity were not confirmed on this study. Conclusions: AD was not declared as the cause of death in the majority of certificates, contributing to the underreporting and reducing the information of death due to AD in the country.", "keywords": [ "Dementia", "Alzheimer disease", "mortality", "nutritional value", "survivorship" ], "content": "Introduction\n\nThe dementias are classified among the most frequent health problems in elderly people1. Alzheimer disease (AD) stands out as the most prevalent2, occurring in more than 50% of the cases3,4.\n\nAlzheimer disease facilitates the advent of opportunistic diseases, which combined to the fragility of the person, deglutition disturbances, and malnutrition can increase the death risks1. People with AD live on average 8.3 years from diagnosis among 65 years old, and 3.4 with diagnosis after 90 years4,5.\n\nAccording to the World Health Organization (WHO), AD can be considered as a cause of death and must be registered on the death certificate of the patients5,6. Nevertheless, in the majority of cases, the main death cause is not related to AD, but other issues are usually listed as immediate causes. According to Ministry of Health the immediate cause of death is the disease, injury or last complication that occurred immediately prior to the moment of death, having a direct correlation with the basic cause of death7.\n\nThe lack of national data still constitutes a gap in the knowledge of the mortality due to AD, although it has a known impact on the patient, family and caregiver.\n\nIt is known that at the present time there is no medical intervention capable of preventing or curing AD, but a protective effect can come from a good diet and quality of life, therefore, nutrition is shown to play an essential role. In this context, the present study aimed to investigate whether nutritional status was associated with AD survival and mortality, and if AD was recorded as the underlying cause of death in the death certificate.\n\n\nMethod\n\nThe present study was conducted in the city of Guarapuava, central-west region of Paraná state, Brazil. We performed a cross sectional study with elderly citizens of the community registered in the National Health System (SUS), through which they receive, at no charge, specific drugs for AD8.\n\nThe patients of this study had their diagnosis of AD performed by SUS doctors, according to the “Neurological and Communicative Disorders and Cerebral Vascular Accident National Institute and the Association of Alzheimer Disease and Related Disturbs (NINCDS – ADRDA)”9.\n\nData were accessed in CELEPAR System (Information and Communication Technology Company of Paraná) after research project approval. After the identification (name and address) of the 66 patients with AD, provided by a computerized system (CELEPAR System), visits were made to patients' homes. Patients / caregivers were invited to take part in the study, and a written informed consent form was signed by a person responsible/patient caregiver who agreed voluntarily to participate in the study.\n\nData collection was carried out in two phases: the first between August and October 2011, for the anthropometric, nutritional measures, and hematologic exams. “Exclusions from the study were as follows: 7 patient due to death, 11 patient because they had moved from the city, 2 were not found at the address registered in the system, and 16 the caregivers did not accept to participate due to the patient status of weakness”. This left 30 patients remaining in the study.\n\nResearch was done in two phases, between August 2011 and June 2013. The first stage was based on collecting nutritional and anthropometric measurements, as well as hematological tests. One and a half years later (June 2013), the researchers contacted the patients or caregivers, verifying that 30% (n=9) of the patients died since the first stage, in 2011. Death certificates (Dc) were evaluated in accordance with International Disease Classification (CID-10)10. It was defined as death due to AD in cases which had AD as the main cause of the death on the death certificate. Death with AD was recorded when it was mentioned in any part of the medical report of the death certificate. It was considered as codes CID G30.0 to G30.910.\n\nWith the intention of evaluating the anthropometrical measures of the patients, we performed a body mass index (BMI). The weight and the height were collected according to the methods advocated in the feed and nutritional surveillance system (SISVAN)10. BMI measurements were taken using a digital precision scale with kilograms scale from Plenna®. To obtain the height values, a stadiometer was used with a centimeter scale. When it was not possible to obtain the weight and height, as some patients could not stay in the orthostatic position since the patients were bedridden, estimated values were used from the formula proposed for Chumlea (1985)11. BMI was calculated using the correlation between the square of total corporal weight (kilograms) and the height (meters), using the cut points for elderly people proposed for Lipschitz (1994)12.\n\nThe nutritional state of the participants was evaluated using the mini nutritional assessment (MAN) (13). The MAN is an instrument composed of measures and practical questions that comprise anthropometric evaluations (weight, arm and calf circumferences, height and loss of weight record), global evaluation (life style, drugs, mobility and diseases), dietary evaluation (qualitative and quantitative) and self-evaluation (nutrition perception). The sum of MAN scores allows discrimination between patients, where values below 17 characterize the patient as undernourished, between 17 and 23.5 at malnutrition risk and between 23.5 and 30 as normal13. The MAN was applied to the patients classified with Clinical Dementia Rating (CDR) 2 or 3. The food consumption was evaluated through the patient or caregiver report of consumed food through memory of 24 hour estimated in home measurements converted to grams14. This data was analysed on Avanutri 4.0® software. Each nutrient was compared to the Dietary Reference Intake (DRI’s)14,15 according to gender and age, due to the lack of specific recommendations for AD patients.\n\nAlso, for the staging of the demential process the Clinical Dementia Rating (CDR) was used. The clinical evaluation of dementia classifies the development stage of AD, where zero rating represents normal, 0.5 dementia questionable (CDR 0.5), 1 mild dementia (CDR 1), 2 moderate dementia (CDR 2) and 3 severe dementia (CDR 3)8.\n\nThe blood collection was performed at patients’ home according to the recommendations for venous blood collection from the Brazilian Society of Clinical Pathology and Laboratory Medicine (2010)16.\n\nThe samples of serum were analysed in biochemistry semi-automated equipment CA 2006 (SHEL – B4B Group, Brazil), and commercial kits (Labtest® - Minas Gerais, Brazil). The total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very low-density lipoprotein (VLDL) cholesterol and triglycerides were evaluated according to the parameters from the Brazilian Cardiology Society, 201317,18. The glucose levels were classified according to parameters from the Brazilian Diabetes Society, 201418. The albumin was dosed through colorimetric methodology, using bromocresol green on the biochemistry semi-automated equipment CA 2006 (SHEL – B4B Group, Brazil), and ALBUMINA PP (Cat# 419, Gold Analisa Diagnostica SA, Brazil) and evaluated following Painter, Cope and Smith parameters19.\n\nThe complete blood count (CBC) was carried out in a hematological analyser Cell-dyn Ruby (Abbott, Illinois, USA) with 33 parameters that use flow cytometry for the white cells, erythrocytes and platelets analysis and the spectrophotometric methodology for the determination of hemoglobin. The hematological parameters were taken from the WHO20.\n\nThe statistical analysis was completed using SPSS package version 20.0. Initially, it was carried out using non-parametric analysis due to the large number of variables that do not fall in a normal distribution when evaluated for Shapiro-Wilk test. The first step of the analysis consisted of the comparison of the initial evaluation (base line) between the subjects who have died during the study, and those revaluated in a later phase according to the test proposed by Mann-Whitney. This step also analyzed the survival according to the tests proposed by Kaplan-Maier and considering CDR, IMC, clinical variables, such as age and, diagnosis data as time variable. This step was also analyzed by Qui-Square test with corrections to verify associations between death and sociodemographic and clinical variables.\n\n\nEthics and consent\n\nThe study was approved by the Research Education Committee of the State University of the Center-West - COMEP / UNICENTRO through process 026/2011. Written informed consent was obtained from a person responsible/patient caregiver for all participants.\n\n\nResults\n\nRespiratory issues accounted for the majority of deaths (Table 1). Alzheimer disease was specified in 22% of cases (n=2) and the patients without a specific cause of death on the death certificate was 22% (n=2). No patient had ICD G30.0, early-onset Alzheimer's disease (Table 1)9.\n\nCID, codes according to the International Disease Classification review (CID -10), DO, death certificate.\n\nAs shown in Table 2, the anthropometric variables and macronutrients ingestion are compared between live and patients who died up until two years after the study evaluation. None of the subjects showed values greater than zero on basophils, atypical lymphocytes, blasts, myelocytes and metamyelocytes variables; values greater than zero indicate pathologies.\n\nP<0,05 †BMI: Body Mass Index; ‡CC: Calf circumference; §MNA: Mini Nutritional Assessment; \|\|PTN: Protein; ¶CHO: Carbohydrate; *LIP: Lipids; ††CT: Total Cholesterol; §§TRIG: Triglycerides. Mann-Whitney U test.\n\nTable 3 shows the differences on micronutrients consumption between the two groups investigated. The only significant difference found between live and deceased patients, was the phosphorus consumption. Figure 1 shows that the phosphorus consumption was lower those who died compared to the live group, although the phosphorus consumption was sufficient for both groups.\n\nGross value (% adequacy). Mann-Whitney U test\n\nMann-Whitney U test.\n\nIn addition, Table 4 shows that there were no deaths in patients classified as eutrophic (MAN specification), while all the undernourished patients and at risk of it had died.\n\nCDR: Clinical Dementia Rating; †MMSE: Mini Mental State; § MNA Examination Mini nutritional Assessment, # Body Mass Index (BMI).\n\nAccording to the CDR classification of the patients who died, 55% were in mild cognitive failure. Of these, 44% were male, which represents half of the male group of the sample. The health conditions showed that 23% (n=7) of the patients had some type of cancer, and from them, 42% (n=3) have died.\n\nFigure 2 shows the AD patient survival curve as a function of age. The survival rate is modified for the disease stage and the results demonstrate a lower survival rate for the patients in the early stages of the disease.\n\nSurvival curve of AD patients distributed by health condition (A) and (B) disease stage. *Kaplan-Meier test.\n\n\nDiscussion\n\nThis study has observed that the respiratory issues may appear as one of the main death cause in AD patients, which may be related to age, due to the advanced age of the majority of the patients. The limitation of the respiratory functional system on AD, due to the depletion of the cough reflex and the accumulation of discharge, reduce the pulmonary ventilation and the elderly immunological system is more affected, facilitating the occurrence of infection, especially respiratory ones21,22.\n\nThe elderly with dementia can exhibit dysphagia, making necessary the use of enteral probes for feeding and the elimination of the discharge from upper airways, increasing the respiratory infections. Pneumonia is one of the most common causes of hospitalization of AD patients and its pathology is directly related to its mortality21–24.\n\nFurthermore, seniors are more likely to present with secondary drug effects, increasing the death risks. The elevated chance of pneumonia in AD patients has to be remembered when treating these people25. A study in Finland compared the risk of pneumonia associated with acetylcholinesterase inhibitors (AChEls) (donepezil, Transdermal rivastigmine, galantamine and memantine) in people with AD diagnosis and concluded that people who used rivastigmine and memantine in comparison with donepezil had an increased risk of pneumonia26. This risk occurred, not only from the drug characteristics, but also for the severity of the AD. The same study considered the use of pneumococcal vaccination for elevated risk of pneumonia with great efficiency26. In Brazil, memantine is used only in moderate and severe stages of the disease.\n\nOther medicines that deserve special attention are the neuroleptics, or tranquilizers, used in AD patients to control the agitation, hostility, delusions and hallucinations26. These medicines are antagonists of dopamine, decreasing its transmission and compromising the deglutination function, facilitating aspiration pneumonia26. The effects of the neuroleptics on the dopaminergic system usually occurs in severe AD, so it is used in lower doses.\n\nOn the other hand, the benzodiazepines that can also be used as tranquilizers in AD have slight effects on the dysphagia, decreasing aspiration pneumonia26.\n\nFor cause of death analysis, the death certificates must be filled in a correct form. The death certificate is the only source of mortality data and depends directly on the quality of the information provided by the doctor27–29.\n\nThe underlying death cause was defined by the World Health Organization as a disease or lesion that had initiated the pathological events chain and brought the patient directly to death6. In line with the methodology proposed for this study, it was recorded as AD with the codes CID G30.0 to G30.9. Underlying cause of death being AD, the code G30.0 did not appear in any certificate, reinforcing the idea that the death certificates frequently register the immediate death causes instead of to register AD as a cause28,29.\n\nAD generally causes undernourishment to the elderly and it can be associated with physiological alterations, dental issues, physical limitations, and diseases that decrease the appetite, food absorption and, drug interactions7,30.\n\nAlthough there is no difference between the groups in this study, it could be observed that values are below those recommended by the Dietary Reference Intakes - DRIS (15) for almost all micronutrients. Figure 1 shows that phosphorus was the only micronutrient with significant differences found between the live and dead group31. However, this difference cannot explain the observed mortality rates, since the percentage of this micronutrient adequacy was inside the normal values in those who died and above the recommended level in the live group7,31. In addition, the most significant symptoms of phosphorus deficiency include bone weaknesses, discomfort in many joints, weakness, dental decay and rickets31,32. In this study, there was no biochemical analysis of phosphorus dosage in the study groups due to the methodology limitations.\n\nTherefore, the early diagnosis of malnutrition is important due the association with the greater mortality in AD. The data from this study supports this, since all the patients who have died were undernourished according to MAN classification7,32.\n\nThe consequences of the weight loss and malnutrition in people with dementia occurs due to the dependency of care, difficulties in mobility, food preparation, chewing and deglutition. Moreover, the nutritional status of the elderly AD patients has a significant impact on the disease course, affecting cognitive parameters and functional symptoms7,30,31.\n\nAdditionally, the malnutrition causes a decrease in white blood cells, leading to alterations in antibodies production and immune resistance, decreasing the survival rate in undernourished patients33. Thus, to reduce or at least delay the AD prevalence, maintaining a proper diet from childhood is crucial and reinforced due the fact that the cardiovascular risks are directly related with the development of dementia.\n\nIn relation to the AD associated factor, our study reveals that 33% of those who died had higher levels of cholesterol and 44% had diabetes. An epidemiological study has demonstrated the directly relation between high cholesterol levels and the increased risk of cognitive issues, vascular alterations and AD34. In terms of diabetes, in this study its presence was not associated with the mortality rates; the survival curve did not show differences between subjects with and without diabetes (p=0.572), although diabetes mellitus is a disease which has risk factors common to AD34.\n\nA prospective study from 2010 has evaluated the associated factors of evolutionary differences and mortality due to AD between males and females. The males had more comorbidities due the differences in exposure, work, alcohol consumption, tobacco consumption and differences on the attitudes related to the disease, increasing the chance of death35, reinforcing our study, where 44% of the those who died during the study were males. In some cases, the male mortality occurs due to external causes, given that females have higher chances of developing AD in advanced ages, correlating with our sample composition27,35. On the other hand, a study conducted by Teixeira et al. (2015) shows a higher mortality in AD females, which may be explained by the prevalence of cases in this gender in Brazil and other countries.\n\nAmong the possible comorbidities occurring in this population, cancer stands out, decreasing survival in the elderly. This effect was not completely observed in this study, where 76.7% of the patients were not cancer patients. From the 23.3% that were cancer patients, 57% have died since the beginning of the study. Still according to Teixeira et al. (2015), the neoplasms were more frequent in patients without AD29.\n\nIn the study performed by Mussico et al. (2013), elderly patients with cancer have a reduced risk of dementia due to AD and vice-versa36. Although demonstrating common pathophysiological mechanisms such as mitochondrial dysfunction, oxidative stress and DNA damage that are propitious to the genetic and metabolic connections between cancer and neurodegeneration, there is an inverse association between the pathologies36,37. Romero (2014), in a prospective study, analyzed the cancer notification proportion on death certificates of dementia patients and concluded that AD patients have a lower risk of mortality due to malignant neoplasia38. In our study, the survival curve has not shown significant differences when the subjects with and without cancer were compared (p=0.998).\n\nA decrease in the survival rate was observed in Figure 2 showed a difference between the distinct disease stages. However, the lower survival values were observed in patients who were classified in the mild dementia stage, an unexpected result that is not in line with the literature6,23,28. Nevertheless, the death of patients of the study is strongly related to respiratory issues and to the progression of the disease. The risk factors for pneumonia on initial AD stage are not sufficiently understood, but one possibility is that a silent aspiration of saliva or bolus without causing cough, respiratory difficulty or other external sign can cause pneumonia due to aspiration24,39. Also, because the symptoms and effects caused by the AD are less evident in mild disease stage patients, the preoccupation with immunological care may be reduced33.\n\nFurthermore, while several studies use the diagnostic time as the survival time, this study has not used it; first due to the difficulties, such as lack of specialized doctor in SUS to assist the great demand and the lack of information about AD, confusing its symptoms with simple senility or not searching for an appropriate diagnostic. Second, the correlation with age is associated with the disease and the survival.\n\nThe obtained results were discussed and interpreted considering the limitation of a cross sectional study with a limited number of patients. In addition, the information was not collected before the patient’s death due to the improbability of each case. Nonetheless, the longest past period since the evaluation and the death was 18 months. Thus, the number and quality of the information fortify the results with social relevance, since they were obtained from a non-institutionalized dementia, where caregivers are relatives, a common situation among people with dementia in Brazil.\n\nThe nutritional status, biochemical and basic comorbidities were not confirmed in this study to explain this relation. Therefore, the AD patients profile characterization in the study and the observed reduction on the survival probability may indicate the absence of proper attention, suggesting new searches for its confirmation. At the same time, the quality of the information in the death certificates that frequently do not register dementia as an implicit cause generates underreporting of the long-term chronic degenerative diseases.\n\nOther limitation is the quality of information given in the death certificate, which does not record AD as an implied reason, giving no notice of chronic degenerative diseases and hiding results. This suggests there is a need for more research in this area, because there is still a shortage of studies on this subject.\n\n\nData availability\n\nAll raw data was freely available on the Open Science Framework site: Doi: 10.17605/OSF.IO/PE38C.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed\n\n\nGrant information\n\nThis study was funded by the Association of studies, research and assistance to people with Alzheimer's disease (AEPAPA), the Araucaria Foundation and Coordination of Improvements of Higher Education Personnel (CAPES).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors would like to extend their gratitude to Association of studies, research and assistance to people with Alzheimer's disease (AEPAPA), to Araucária Foundation and Higher Education Personnel Improvement Coordination (CAPES).\n\n\nReferences\n\nAlzheimer's Association: 2011 Alzheimer’s disease facts and figures. Alzheimer Dement. 2011; 7(2): 208–44. PubMed Abstract \| Publisher Full Text\n\nAprahamian I, Martinelli JE, Yassuda MS: Doença de Alzheimer: revisão da epidemiologia e diagnóstico. Rev Bras Clin Med. 2009; 7: 27–35. Reference Source\n\nPrince M, Guerchet M, Prina M: The Global Impact of Dementia 2013–2050. London: Alzheimer's Disease International. 2013; 8. Reference Source\n\nWimo A, Winblad B, Jönsson L: The worldwide societal costs of dementia: estimates for 2009. Alzheimers Dement. 2010; 6(2): 98–103. PubMed Abstract \| Publisher Full Text\n\nMendonca FM, Drumond E, Cardoso AM: Problemas no preenchimento da Declaração de Óbito: estudo exploratório. Rev Bras Estud Popul. 2010; 27(2): 285–95. Publisher Full Text\n\nFerreira AB, Ribeiro FF, Fontenele RP, et al.: Mortalidade pela Doença de Alzheimer no Brasil entre os anos de 2000 a 2013. Acta Ciênc & Saúde. 2015; 1(1): 100–115. 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PubMed Abstract \| Publisher Full Text\n\nSociedade Brasileira de Patologia Clínica/Medicina Laboratorial BPC/ML: Recomendações da Sociedade Brasileira de Patologia Clínica para Coleta de Sangue Venoso. 2th ed. Barueri, SP, Minha Editora. 2010.\n\nMalachias MV, Souza WK, Plavnik FL, et al.: 7ª diretriz brasileira de hipertensão arterial. Arq Bras Cardiol. 2016; 107(3): 370. Reference Source\n\nSociedade Brasileira de Diabetes: Diretrizes da Sociedade Brasileira de Diabetes 2013–2014. São Paulo: AC Farmacêutica, 2014.. Reference Source\n\nPainter PC, Cope JY, Smith JL: Reference information for the clinical laboratory.In: Burtis CA, Ashwood ER eds. Tietz textbook of clinical chemistry. Philadelphia: WB Saunders Company, 1999;477–540.\n\nWorld Health Organization, DeMaeyer EM, Dallman P, et al.: Preventing and controlling iron deficiency anaemia through primary health care: a guide for health administrators and programme managers. Geneva. 1989. Reference Source\n\nHendriks SA, Smalbrugge M, van Gageldonk-Lafeber AB, et al.: Pneumonia, Intake Problems, and Survival Among Nursing Home Residents With Variable Stages of Dementia in the Netherlands: Results From a Prospective Observational Study. Alzheimer Dis Assoc Disord. 2017; 31(3): 200–208. PubMed Abstract \| Publisher Full Text\n\nLarson, EB, Shadlen MF, Wang L, et al.: Survival after initial diagnosis of Alzheimer disease. Ann Intern Med. 2004; 140(7): 501–9. PubMed Abstract \| Publisher Full Text\n\nBonsignore M, Heun R: Mortality in Alzheimer’s disease. Dement Geriatr Cogn Disord. 2003; 15(4): 231–36. PubMed Abstract \| Publisher Full Text\n\nWada H, Nakajoh K, Satoh-Nakagawa T, et al.: Risk factors of aspiration pneumonia in Alzheimer’s disease patients. Gerontology. 2001; 47(5): 271–276. PubMed Abstract \| Publisher Full Text\n\nLampela P, Tolppanen AM, Tanskanen A, et al.: Use of antidementia drugs and risk of pneumonia in older persons with Alzheimer’s disease. Ann Med. 2016; 49(3): 230–39. PubMed Abstract \| Publisher Full Text\n\nZagozdzon P, Goyke B, Wrotkowska M: Mortality Rates in Users of Typical and Atypical Antipsychotics: A Database Study in Poland. Drugs Real World Outcomes. 2016; 3(3): 345–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOtero UB, Rosenfeld S, Gadelha AJ: Óbitos por desnutrição em idosos em São Paulo e Rio de Janeiro: análise de séries temporais, 1980–1986. Rev Bras Epidemiol. 2001; 4(3): 191–205.\n\nJames BD, Leurgans SE, Hebert LE, et al.: Contribution of Alzheimer disease to mortality in the United States. Neurology. 2014; 82(12): 1045–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBonini JS, Goes VF, Oliveira CF, et al.: Psychophysiological, cognitive and behavioral aspects of malnutrition in Alzheimer’s disease: a review. Rev Bras Ciênc Envelhecimento Humano. 2014; 11(3): 245–56. Publisher Full Text\n\nHernando-Requejo V: Nutrition and cognitive impairment. Nutr Hosp. 2016; 33(Suppl 4): 346. PubMed Abstract \| Publisher Full Text\n\nAndrási E, Páli N, Molnár ZS, et al.: Brain aluminum, magnesium and phosphorus contents of control and Alzheimer-diseased patients. Am J Alzheimers Dis. 2005; 7(4): 273–84. PubMed Abstract \| Publisher Full Text\n\nSchäufele M, Bickel H, Weyerer S: Predictors of mortality among demented elderly in primary care. Int J Geriatr Psychiatry. 1999; 14(11): 946–56. PubMed Abstract \| Publisher Full Text\n\nLopes da Silva S, Vellas B, Elemans S, et al.: Plasma nutrient status of patients with Alzheimer’s disease: systematic review and meta-analysis. Alzheimers Dement. 2014;10(4): 485–502. PubMed Abstract \| Publisher Full Text\n\nKivipelto M, Helkala EL, Hänninen T, et al.: Midlife vascular risk factors and late-life mild cognitive impairment: A population-based study. Neurology. 2001; 56(12): 1683–9. PubMed Abstract \| Publisher Full Text\n\nDriver JA: Inverse association between cancer and neurodegenerative disease: review of the epidemiologic and biological evidence. Biogerontology. 2014; 15(6): 547–57. PubMed Abstract \| Publisher Full Text\n\nSinforiani E, Citterio A, Zucchella C, et al.: Impact of gender differences on the outcome of Alzheimer's disease. Dement Geriatr Cogn Disord. 2010; 30(2): 147–54. PubMed Abstract \| Publisher Full Text\n\nMusicco M, Adorni F, Di Santo S, et al.: Inverse occurrence of cancer and Alzheimer disease: a population-based incidence study. Neurology. 2013; 81(4): 322–328. PubMed Abstract \| Publisher Full Text\n\nRomero JP, Benito-León J, Louis ED, et al.: Alzheimer's disease is associated with decreased risk of cancer-specific mortality: a prospective study (NEDICES). J Alzheimers Dis. 2014; 40(2): 465–73. PubMed Abstract \| Publisher Full Text" }	[ { "id": "31762", "date": "12 Mar 2018", "name": "Robert Stewart", "expertise": [ "Reviewer Expertise Dementia epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report is of a small study of 30 people with Alzheimer's disease, 9 of whom died within 1.5 years of baseline assessment. Information is provided on recorded cause of death and participants who died are compared to the remainder on a range of anthropometric, haematological, biochemical and nutritional indices recorded at baseline.\nI'm all in favour of data being in the public domain; however, the discussion section needs to be fundamentally revised before the paper can be viewed as accepted. The conclusions which can be drawn are very limited by the sample size and do not adequately support the extensive material presented in the discussion, so this should be substantially abbreviated as described below. There are also aspects of the methodolgy which need to be clearer.\nWhile it is reasonable to display a breakdown of causes of death, and reasonable to comment on the relatively low proportion of death certificates mentioning AD (which has been described in other samples), it is not appropriate to draw conclusions about dementia-related factors contributing to causes of death. Conditions such as respiratory disease are common causes of death in any older population and the study does not evaluate whether they are any higher in people with dementia than the general population, so it is misleading to be discussing, for example, elevated risks of pneumonia or cancer or whatever.\n\nAlthough I don't object to the authors displaying descriptive data on their baseline covariates stratified according to subsequent death, a study of this size would only be able to identify very large differences at statistical significance; therefore I don't feel that it is appropriate to be discussing the findings in any more detail than a simple presentation of descriptive data. The difference in phosphorus levels is highly likely to represent type 1 statistical error because of the very large number of comparisons made, so it is reasonable not to draw any inferences from this. The rest of the discussion about risk factors for mortality is not justified because the study does not have statistical power to detect these associations and, indeed, no significant associations are identified.\n\nIt is reasonable to comment on levels of survival in the sample, but I don't think that anything can be concluded about stage of dementia and survival because the numbers are simply not large enough.\n\nThe repeated description of the study as cross-sectional is misleading. The study took a group of 30 people and followed them up for mortality. It is therefore surely a cohort study? Similarly, I don't understand the sentence in the third-to-last paragraph: \"In addition, the information was not collected before the patient’s death due to the improbability of each case.\" Surely all the information was collected before death (apart from the death certification)?\n\nAs far as I can tell, the participants were people who were recorded as receiving medications for AD and who were then approached and visited at home. There does not appear to have been any formal evaluation of the diagnosis, apart from the completion of a CDR. Is this correct? It would be helpful if the sample identification and recruitment process was a little more clear.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "30464", "date": "04 Sep 2018", "name": "Irving E Vega", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors addressed two important aspects related to Alzheimer's disease: 1) accuracy of mortality rates, and 2) nutrition as a risk factor. However, the experimental design presented in the current version of the manuscript fall short in addressing both issues. Several important shortcomings in the experimental design and interpretation of the results render this manuscript unacceptable. For example:\nThe authors compare or use different variables in the study but does not present a power analysis to support that the number of participant is sufficient to draw any conclusion.\n\nIt is unclear how nutritional status relate to the death certificate. The introduction is confusing as to what is the gap in knowledge and the relationship of the proposed questions.\n\nThe main limitation of death certificate is that, in the case of AD, the diagnosis is not confirmed (i.e. neuropathology).The authors did not address this major limitation.\n\nWho completed the MAN? A caregiver? How this data was collected?\n\nCancer patients need to be segregated from the data since may have contributed to the only difference observed; phosphorus level.\n\nThe participants are not segregated based on stage of AD (i.e. mild, moderate, severe). It is expected that the stage of the disease will affect their nutritional level.\n\nThe discussion section addressed issues not supported by the data or even related to it. For example: Tthere is no data on the use of any of the drugs mentioned by the research participants. How is this information relevant to the study?\n\nIn summary, in the present form, the authors do not have any supporting data for the interpretations and conclusion presented in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-137
https://f1000research.com/articles/7-136/v1	01 Feb 18	{ "type": "Research Article", "title": "Alexiteric activity of Costus pulverulentus C. Presl., Desmodium adscendens (Sw.) DC., Begonia glabra Aubl. and Equisetum bogotense on the poison of Bothrops asper (equis)", "authors": [ "Wilson Tapia", "Katy Garzón", "Nelly Granda", "Bence Mátyás", "Wilson Tapia", "Katy Garzón", "Nelly Granda" ], "abstract": "Background: According to the Ecuadorian ancestral knowledge, antivenom treatment can be carried out using medicinal plants; there are in vitro studies in which alexiteric activity has been proven in many plant species through the assay of reduction of indirect hemolytic activity in blood agar plate-phosphatidylcholine. The cases of snake bites \"equis\" on the Ecuadorian coast represent the largest number of reported ophidian accidents, which usually occur in remote areas of health centers or hospitals, making patient care not timely. The species Costus pulverulentus C. Presl, Desmodium adscendens (Sw.) DC., Begonia glabra Aubl., and Equisetum bogotense are considered plants with antivenom potential (alexiteric) and have been used by various Ecuadorian ethnicities. Methods: In the present investigation, the indirect anti-hemolytic activity in vitro of dry extracts of these four-plant species was evaluated, as an indicator of alexiteric activity, on three \"pools\" of Bothrops asper (equis) snake venom provided by the Vivarium of Quito (Quito, Ecuador).\n\nResults: Alcoholic extracts of the four-species showed neutralizing activity of the venom, with some differences according to the treatment and origin (composition) of the venom. The aqueous extract of B. glabra presented antihemolytic activity, unlike the other species. Conclusions: We conclude that the alcoholic extracts of the four-species presented activity with some differences according to treatment and origin (composition) of the poison, and the aqueous extract of B. glabra presented anti-hemolytic activity unlike the other species whose activity was null.", "keywords": [ "Bothrops asper alexiteric activity", "Desmodium adscendens", "Begonia glabra", "Costus pulverulentus." ], "content": "Introduction\n\nOphidic accident is defined as injury resulting from the bite of a poisonous snake that causes toxic effects on the victim. Snake venom is a complex mixture of protein-enzymatic substances and non-protein substances that produce pain, edema, and drop in blood pressure, hemolysis, proteolysis and alterations in blood coagulation1. It can be considered a public health problem both by the effects they generate and by the complications derived from inopportune treatment, causing disability or even death of the patient.\n\nIn Ecuador, 1953 cases were reported during the year 2016 and 313 cases have been reported between January and March 20172. In the littoral region, the most common species is Bothrops asper (equis), while Bothrops atrox (pitalala) is the most common in the Amazon region3.\n\nIn traditional medicine, many plants have been used to combat the effects of venomous snake bites; they are called alexiteric because they are used to mitigate the effects of the venom of poisonous animals4. Indirect anti hemolytic activity of Equisetum bogotense Kunth has shown a reduction in the hemolytic effect induced by B. asper venom5. The species Costus pulverulentus C. Presl, Desmodium adscendens (Sw.) DC., Begonia glabra Aubl. and Equisetum bogotense, listed in the book \"Useful Plants of Ecuador\"6, are considered plants with antiophidic potential.\n\nThe present study evaluated the anti-hemolytic effect in vitro, used as an indicator of alexiteric activity, of these plant species to contribute in the search for solutions to the problem triggered by ophidian accidents.\n\n\nMethods\n\nThe plant samples were collected in areas of northwestern Pichincha (Table 1) and species were identified at the National Herbarium of Quito (Quito, Ecuador).\n\nThe parts used for the plant species, according to the traditional use of the inhabitants of the area, to be tested are as follows:\n\nCostus pulverulentus C. Presl.: rhizomes\n\nDesmodium adscendens (Sw.) DC.: whole plant\n\nBegonia glabra Aubl.: leaves\n\nEquisetum bogotense Kunth.: whole plant\n\nPlant samples were dried in an oven at 37 degree C for 48 h, and then were ground to a particle size of 5 mm from which extracts were prepared by maceration with solvents of increasing polarity (n-hexane < alcohol < water). Hexane extract: 25 g of dried plant material was macerated with 350 mL of n-hexane at room temperature for 48 h. Then it was filtered and concentrated to dryness in rotary evaporator at 40 degree C and stored in an amber vial. The dry residue of this extraction was used to obtain the following alcoholic extract. Alcoholic extract: The dry residue from the previous extraction was macerated with 350 mL of absolute ethyl alcohol at room temperature for 48 h. Then it was filtered and concentrated to dryness in rotary evaporator at 40 degree C and stored in an amber vial. The dry residue of this extraction was used to obtain the following aqueous extract. Aqueous extract: The dry residue from the previous extraction was macerated with 350 mL of distilled water at room temperature for 48 h. Then it was filtered and concentrated to dryness in a water bath at 40 degree C and stored in an amber vial.\n\nThe sample of poison was obtained by manual milking of specimens in captivity (Figure 1), carried out by the staff of the Herpetological Foundation \"Gustavo Orcés\" (Vivarium of Quito, Quito, Ecuador). These specimens corresponded to three \"pools\" of B. asper venom from of the Ecuadorian coast, according to the specifications in Table 2.\n\nThe pools of venom were stored in a vacuum desiccator protected from light and refrigerated at 4°C until the tests were carried out in the laboratories of the Life Sciences Area of the Universidad Politécnica Salesina, Campus Giron- Quito.\n\nThe technique described in 7 was followed. Briefly, on a Petri dish containing blood-phosphatidylcholine agar that was prepared by mixing 1% agar, 0.1 mm CaCl2, 0.33% egg yolk and 1.2% washed erythrocytes8, 15 µL of each dilution of venom, in physiological saline (4.76%, 2.44%, 1.23%, 0.62% v/v), was sown. The petri dishes were incubated for 20 h at 37°C. The halo of hemolysis was measured in mm. The lowest dose of venom that generated a 10 mm halo was taken as MHD.\n\nThe technique described in 8 was followed. Briefly, plant extract (1.6µg/mL) was pre-incubated with the MHD of the ophidian venom (ratios of 1: 7; 1:10; 1:20; poison: extract) for 30 minutes at 37°C. µL. 15 µL of this mixture was plated on a 6 mm-diameter petri plate containing blood-agar-phosphatidylcholine and incubated for 20 h at 37°C. The test was carried out in triplicate for each extract and for each concentration.\n\nAnalysis of variance was performed and after verifying significant differences, the Tukey test was applied using the Infostat/L program, version 11.1.2014, with a significance of 95%.\n\n\nResults\n\nTable 3 shows the values obtained for the minimum dose hemolytic (MIDH) poison of B. asper from different areas of the Ecuadorian coastal region. It is shown that pool V2, from Esmeraldas an Manabí province, presents greater hemolytic activity compared with V1 and V3, from the Northwest of the Pichincha and Bolivar provinces, respectively. These two pools have the same activity; however, must consider that the three poisons are mixtures obtained from specimen captive males and females, adults and young from each of the above locations.\n\nExample photographs of the MHD can be seen in Supplementary File 1.\n\nTable 4 shows the results, assigning the sign (+), for the treatment that reduced the diameter of hemolysis produced by the MHD of each pool by 100%. The alcoholic extract of E. bogotense has very good inhibitory activity of the hemolytic effect produced by the venom of B. asper, which has also been shown in previous research on Bothrops atrox (Yarlequé amp; et al., 2012). However, in the case of B. glabra, its aqueous extract as well as its alcoholic extract is also effective. This shows that this plant is the most effective species of all tested.\n\n((++) is 100% inhibition; (+) is lower activity; (-) is zero activity).\n\nThe alcoholic extracts of E. bogotense and D. adscendens present antihemolytic activity in all treatments and for the three pools of venom, which shows they are the most active species against the hemolytic effect of Bothrops asper venom. Neither the aqueous nor the hexane extract of these two species demonstrate this activity.\n\n\nConclusions\n\nThe aqueous and alcoholic extracts of B. glabra show antihemolytic activity for all the treatments. In particular, the aqueous extract is effective against the anti-hemolytic effect of V1 and V2 and the alcoholic only against V2. However, no extract of B. glabra demonstrated efficacy against the V3 pool. The species with the least alexiteric activity is C. pulverulentus, although it is active in front of the pool V1.\n\nAlthough there are differences in activity according to extracts, plant species and origin of venom, the four evaluated plant species could be used in the prevention of the symptoms of a bothrophic accident. Taking into account their ancestral use, namely their consumption, this may be a preventive measure against the effects of Bothrops asper venom in the absence of adequate and timely professional medical treatment.\n\n\nEthical statement\n\nAll efforts were made to ameliorate any suffering to the animals following the protocol described by the World Health Organization9 in accordance with the Ecuadorian code of practice for the care and use of animals for scientific purposes of the Ministry of Environment of Ecuador. Our experiments were approved by the Ministry of Environment of Ecuador (research permit number: No. 07-2014-IC-FAU-FLO-DPAP-MA).\n\n\nData availability\n\nDataset 1. The minimum hemolytic dose raw data for the four plant species against the three pools of venom. The antihemolytic activity percentages for all extracts of plant species, and concentrations of the three venom pools. DOI, 10.5256/f1000research.13528.d19150110\n\nDataset 2: Raw data for the MHD for the three pools of venom. DOI, 10.5256/f1000research.13528.d19150211", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: Examples of photographs of the results. MIDH in V1, MIDH in V2, MIDH in V3, Treatment V1-extract aqueous E. bogotense Kunth., Treatment V2-extracto aqueous D. adscendens (Sw.) DC., Treatment V3-extract aqueous C. pulverulentus C. Presl, Treatment V1-extracto alcoholic E. bogotense Kunth., Treatment V2-extracto alcoholic D. adscendens (Sw.) DC., Treatment V3-extract alcoholic de C. pulverulentus C. Presl, Treatment V1-extract hexane B. glabra Aubl., Treatment V2-extract hexane de B. glabra Aubl., Treatment V3-extract hexane E. bogotense Kunth.\n\nClick here to access the data.\n\n\nReferences\n\nPineda D: Accidentes por animales venenosos. Bogotá: Instituto Nacional de Salud. 2006.\n\nCueva, Zamora X: Mordedura de serpientes. SMordedura de serpientes. Quito: Universidad Central del Ecuador. 2010.\n\nMinisterio de Salud Pública del Ecuador: Manual de normas y procedimientos sobre prevención y tratamiento de accidentes ocasionados por mordedura de serpientes. 2008. Reference Source\n\nLópez J: Plantas alexitéricas. Antídotos naturales contra la picadura de serpientes venenosas. Medicina naturista. 2009; 17–24. Reference Source\n\nBarranco W: Evaluación biológica preliminar de extractos vegetales utilizados en la medicina natural en ls Sierra Nevada de Santa Marta contra el veneno de Bothrops asper. Duazary. 2012; 140–150. Reference Source\n\nRíos M: En Plantas útiles del Ecuador. Quito: Abya-yala. 2007. Reference Source\n\nCamargo FJ, Torres AM, Tressens SG, et al.: Inhibición de la actividad hemolítica del veneno de Bothrops neuwiedi diporus Cope (yarará chica) por extractos de plantas del nordeste argentino. Corrientes: Universidad Nacional del Nordeste. Obtenido de Comunicaciones científicas y tecnológicas. 2005. Reference Source\n\nCamargo F, Dellacassa E, Ricciardi A, et al.: SDS-PAGE una herramienta útil en la evaluación preliminar de actividad alexítera de extractos vegetales. Boletin latinoamericano y del caribe d eplantas medicinales y aromáticas. 2011; 10(5): 429–434. Reference Source\n\nWorld Health Organization: WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins. World Health Organization, 20 Avenue Appia, 1211 Geneva 27. 2016. Reference Source\n\nTapia W, Garzón K, Granda N, et al.: Dataset 1 in: Alexiteric activity of Costus pulverulentus C. Presl., Desmodium adscendens (Sw.) DC., Begonia glabra Aubl. and Equisetum bogotense on the poison of Bothrops asper (equis). F1000Research. 2018. Data Source\n\nTapia W, Garzón K, Granda N, et al.: Dataset 2 in: Alexiteric activity of Costus pulverulentus C. Presl., Desmodium adscendens (Sw.) DC., Begonia glabra Aubl. and Equisetum bogotense on the poison of Bothrops asper (equis). F1000Research. 2018. Data Source" }	[ { "id": "31166", "date": "07 Mar 2018", "name": "Juan J. Calvete", "expertise": [ "Reviewer Expertise Snake venomics and antivenomics" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors should provide an overview of the major life-threatening effect of an Ecuadorian bothropic snakebite. Major components of Bothrops snakes, particularly B. asper and B. atrox are myonecrotic and immflamatory phospholipase A2s, hemorrhagic metalloproteases, and thrombin-like enzymes, whose substrates include components of the blood clotting system such as factors XII, X, and fibrinogen. What is the relevance of venom-induced hemolysis? This should be clarified, and, when available, the hemolytic toxins briefly described.\nTable 4: The results of the reduction in the hemolytic effect of different mixtures of various plant species extracts pre-incubated with the MHD of the B. asper venom (ratios of 1: 7; 1:10; 1:20; poison: extract) should be better quantified (-, +, and ++ is too vague..).\n\"The cases of snake bites \"equis\" on the Ecuadorian coast represent the largest number of reported ophidian accidents, which usually occur in remote areas of health centers or hospitals, making patient care not timely\". Authors conclude that \"the four evaluated plant species could be used in the prevention of the symptoms of a bothrophic accident\". However, and given that the authors propose \"the consumption of the extracts sampled as a preventive measure against the effects of Bothrops asper venom in the absence of adequate and timely professional medical treatment\", the in vitro results should be supported by in vivo experimentation. Without these results, any conclusion is purely speculative and the recommendation of using these plant extracts can be more harmful than beneficial (by giving false confidence to the victim of a snake bite, thereby relegating more an appropriate but expensive medical treatment).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "33719", "date": "16 May 2018", "name": "Andre Lopes Fuly", "expertise": [ "Reviewer Expertise toxinology", "natural products", "coagulation", "platelet aggregation" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript reports inhibitory effects of plants against hemolysis induced by the venom of Bothrops asper snakes, and it postulates the use of plants as antivenom. However, the results are too preliminary to mention that such plants could be used to treat snake bites, since envenomation by Bothrops species produces a myriad of toxic effects such as tissue necrosis, edema and hemorrhage. At the least, more activities should be tested instead of only one; the authors choose an in vitro activity, the indirect hemolytic test, which is not the best to perform in order to postulate a molecule as antivenom. Some in vivo tests must be performed as well to strengthen the manuscript.\nThus, I must reject manuscript as it is presented.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-136
https://f1000research.com/articles/6-66/v1	23 Jan 17	{ "type": "Method Article", "title": "Blockchain protocols in clinical trials: Transparency and traceability of consent", "authors": [ "Mehdi Benchoufi", "Raphael Porcher", "Philippe Ravaud", "Raphael Porcher", "Philippe Ravaud" ], "abstract": "Clinical trial consent for protocols and their revisions should be transparent for patients and traceable for stakeholders. Our goal is to implement a process allowing the collection of patients’ informed consent, which is bound to protocol revisions, storing and tracking the consent in a secure, unfalsifiable and publicly verifiable way, and enabling the sharing of this information in real time. For that, we will built a consent workflow using a rising technology called Blockchain. This is a distributed technology that brings a built-in layer of transparency and traceability. Additionally, it removes the need for third parties, and gives participative control to the peer-to-peer users. From a more general and prospective point of view, we believe Blockchain technology brings a paradigmatical shift to the entire clinical research field. We designed a Proof-of-Concept protocol consisting of time-stamping each step of the patient’s consent collection using Blockchain; thus archiving and historicising the consent through cryptographic validation in a securely unfalsifiable and transparent way. For each revision of the protocol, consent was sought again. We obtained a single document, in a standard open format, that accounted for the whole consent collection process: timestamped consent status with regards to each version of the protocol. This document cannot be corrupted, and can be checked on any dedicated public website. It should be considered as a robust proof of data. In the future, we think that the complex data flow of a clinical trial can be tracked using Blockchain. Moreover, a blockchain core functionality, named Smart Contract, can help prevent clinical trial events not to happen in the right chronological order: including patients before they consented or analysing case report forms data before freezing the database. This will help reaching reliability, security, and transparency, and could be a consistent step towards reproducibility.", "keywords": [ "clinical trial", "blockchain", "smart contract", "consent", "e-consent", "transparency", "reliability", "reproducibility" ], "content": "Introduction\n\nPatient participation is the sine qua non condition for clinical trials to happen and for medical research to improve1,2 (http://www.mc.vanderbilt.edu/crc/workshop_files/2011-09-09.pptx). However, in practice, the informed consent process is hard to handle in a rigorous and satisfactory way. The US Food and Drug Administration (FDA) reports some metrics that are related to the frequency of clinical investigator-related deficiencies, which show that almost 10% of trials they monitored suffer from consent collection issues, such as failure to re-consent when new information becomes available, use of expired forms or non-validated, unapproved forms, consent document not signed or not dated, missing pages in consent document provided to subjects, failure to obtain written informed consent, parental permission obtained after child assent, changes made to the consent documents by hand and without IRB approval3,4 (http://www.fda.gov/downloads/AboutFDA/CentersOffices/CDER/UCM256376.pdf; http://www.yale.edu/hrpp/education/documents/CommonProblemsinInformedConsent_2013_vF.pptx). The study by Seife5 analysed hundreds of clinical trial FDA inspection documents, covering the 1998–2013 period, and showed that a substantial number of them presented evidence of research misconduct, among which 53% were related to failure to protect the safety of patients and/or issues with oversight or informed consent.\n\nThis can lead to dramatic events, as in France in January 2016: a trial testing the “BIA 10-2474” as an analgesic molecule caused the death of a subject. The French health agency IGAS appeared to prove that re-consent was not sought after major neurological side effects occurred in some patients, leading to subjects being included in the clinical trial without being informed of this issue and still receiving doses of the analgesic molecule, (http://www.liberation.fr/france/2016/02/04/drame-de-l-essai-clinique-a-rennes-le-deces-reste-inexplique_1431074; https://fr.wikipedia.org/wiki/BIA_10-2474, version of 2016.09.05). Another example in the UK occurred when a general practitioner was struck off after testing drugs on patients who did not give their consent6. A recent, popular case of serious scientific misconduct was the DECREASE studies performed by Don Poldermans. The results of these studies were invalidated and some related publications retracted as, amongst many other frauds including data invention, informed consent was not proved to have been obtained before the randomised controlled trials (https://en.wikipedia.org/wiki/Don_Poldermans, version of 2016.09.05; http://retractionwatch.com/category/by-author/don-poldermans/).\n\nObtaining an individual’s consent is strictly tied to the Helsinki declaration7,8 (http://www.wma.net/en/30publications/10policies/b3/index.html), which provides the good practices that should follow any stakeholder conducting a clinical trial. Point 26 of the Declaration states that each subject should be informed of the aim, methods, sources of funding, conflict of interests, affiliations of the researchers, anticipated benefits and risks and post-study provisions, and these conditions must be met to obtain freely-given informed consent. In practice, regulation agencies, such as the FDA, provide recommendations and mandatory commitments for consent to be collected in the right conditions9 (http://www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM405006.pdf). Among those and of major importance, informed consent should be documented by a signed and dated written consent, which is particularly meaningful with Blockchain technology.\n\nIn addition, consent collection is a dynamic process; it is not a one-shot process ending when consent is sought before a clinical trial begins. As explained by Gupta10, there are circumstances under which consent has to be sought again from a subject, corresponding to any time the trial protocol is majorly revised. This is a fundamental fact when ensuring to patients’ rights and transparency of a clinical trial11,12. Indeed, as detailed in these Institutional review board (IRB) guidelines (http://www.irb.pitt.edu/sites/default/files/reconsent guidance.pdf; http://www.mayo.edu/research/documents/29-re-consent-or-notification-of-significant-new-findingspdf/doc-10027714; http://www.yale.edu/hrpp/policies/documents/Reconsentingguidance.pdf), there are many situations where patients re-consent has to be sought or where they should be notified of minor trial issues, such as novel risks, significant changes in the research procedures, and worsening of the medical condition. Documents that are to be sent to patients can be consent form addendums, an information letter or a fully revised consent form. Of course, the revised consent form has to be approved by an IRB. It must be stressed that the FDA has highlighted the necessity to conceive a mechanism that ensures that the most recent revised consent form is in use in a clinical trial, and stipulates that timestamping is such an approved mechanism9 (http://www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM405006.pdf).\n\nAs indicated in Figure 1, consent is a dynamic process that involves a complex circuit of data and interacting actors, which should all retain information of this on-going process. For example: what subjects were notified; when the notifications were delivered; whom of the subjects consented or re-consented; when did subjects consent or re-consent. This information should circulate between the clinical trial stakeholders in real time.\n\nBlockchain is a dawn-aged technology, a giant public datastore, stored in a secure and decentralised way. It is widely announced to be a backbone of the circulation of digital assets, powering any kind of services by transparency, traceability. In this context, Blockchain’s emerging and promising technology (https://en.wikipedia.org/wiki/Blockchain_(database)) can bring solid basis in the enrolment phase transparently to all stakeholders of a clinical trial, especially in the context of obtaining participant’s consent. Three core functional principles of this technology can play a fundamental role, as follows:\n\n1. Unfalsifiable timestamping information: this stands for proof of existence of any piece of data. When stored, this data is provable and immutable through a strong cryptographic protocol. Moreover, these proofs of existence can be checked on a public website. This transparency is of interest to any interested stakeholder.\n\n2. Smart contract: this is a contract that is algorithmically implemented and binds any change in the protocol to the patients’ consent seeking renewal.\n\n3. The decentralized nature of the Blockchain protocol gives to the patient, or more widely to patients’ communities, control over their consent and its revocation. The end-to-end connectivity creates a network that empowers patients and researchers as peers.\n\nAt a clinical trial level. Blockchain technology can act as a SafeGuard for the complex and wide range of actors required in clinical trials. In practice, the proof of existence for consent will be timestamped and stored in Blockchains, enabling clinical research stakeholders, such as sponsors, investigators and IRBs, which can be numerous in multi-center clinical investigations, to share consent and re-consent related data in real-time, and archive and historicize consent sets, which can be matched with each revision of the protocol.\n\nObtaining consent must be a ‘lock’ before subject inclusion in clinical trials. Indeed, with the help of Blockchain cryptographic keys, investigators won’t be able to include a patient in the trial until their consent is collected. In addition, to ensure a strict parity between enrolled patients and included patients, the present study will use one core functionality that Blockchain enables: the Smart Contract (https://en.wikipedia.org/wiki/Smart_contract, version of 2016.09.05). This is a piece of code that holds a programmatically written contract between as many parties as needed, without any third-party, which executes algorithmically according to the terms provided by the contracting parties. This makes it possible to build a Smart Contract that will be executed with the only condition that patients will only be included when the enrolment is complete (technically, every Blockchain transactions can have a lock associated to them and transactions can be pending and triggered at an agreed upon contract time).\n\nAt a patient level. Implementing a “privacy by design” technology, and archiving securely and transparently any dataset that needs to be stored, is a game changer towards improving enrolment phase methodology. Moreover, drawing on an entirely new way to collect informed consent, being careful with subjects rights, and so empowering them, could improve the enrolment rate. Indeed, the participation rate to clinical trial remains quite weak11. Caldwell & All performed a systematic review comparing different enrolment techniques, and showed that, amongst several other explanations, the conditions of collecting patients consent in an open and secure way is better at achieving an improved rate of enrolment11,13. At the patient community level, much literature documents barriers to enrolment, especially when barriers are strongly related to community or ethnicity-related issues14,15. We think that the decentralized, transparent and secure nature of Blockchain protocol may meet the conditions for an improved engagement of patient communities in clinical trials.\n\nThe tools that we will present in the current study should be considered as a starting point to define a gold-standard of an open and secure informed consent collection process. This can help optimize patients enrolment, and in turn, through a more transparent and trusted process, can create a bridge between clinical research teams and patient communities, who are novel incomers in our digital age and whose commitment is critically dependent on building clinical trials as a highly trusted process.\n\n\nMethods\n\nIn this proof-of-concept (POC) study, we targeted 27 volunteers for enrolment, which was achieved at the Clinical Epidemiology Department at Hospital Hôtel Dieu (Paris, France). There were no exclusion or inclusion criteria; the volunteers that were recruited were staff members of the Epidemiology Department (Hospital Hôtel Dieu). However, we ensured each of the users had email access.\n\nA fake experimental clinical trial protocol was designed whose purpose was to compare the effect of “cisplatin vs. ledgerlin”, the last substance being a neologism, which was derived from the critical public datastore shared by all Blockchain users called “ledger\". The protocol was accompanied by a consent form, which mimicked a design used routinely.\n\nEach of the to-be-enrolled users were assigned a private key in order to sign data and documents, and in practice this would be used to publish their signed consent, which was to be anchored to the Blockchain.\n\nThere are several Blockchain networks, for example Ethereum (https://www.ethereum.org), Bitcoin (https://en.wikipedia.org/wiki/Bitcoin_network) and Hyperledger (https://www.hyperledger.org). For our purpose, transactions and their validations were run on the Bitcoin network. Our choice was motivated by stability and immutability of the Bitcoin Blockchain, due to its large mining network, and the API it provides facilitates development. Moreover, it is the more widely used Blockchain network; therefore, there is a relatively dense community of developers to enable an efficient support (https://bitcoin.stackexchange.com). The front-end and back-end technologies that are detailed hereafter were implemented by a Blockchain solutions provider, Stratumn SAS (https://stratumn.com/).\n\nA website was developed with a simple one-page interface (Figure 2). On the front-end, it displayed the consent document, a checkbox attesting that the protocol was read and understood, and a push button that triggered the consent process.\n\nIn practice, the on-line signed document contained a piece of code called “Chainscript”, (http://chainscript.io), which contained all the critical information about the user, and the version of the protocol attached to the signature. Each proof of signature had a manifest that takes the form of a hash that is the digital proof of signature.\n\nOn the back-end, pressing the “consent button” triggers Blockchain transactions: the proof of signature is timestamped and stored in the Blockchain. It should be emphasised that these signatures were shared in real-time through a restricted group of individuals, namely the present authors, who stand, in a real-life implementation, for investigators, IRBs or sponsors. This group obtains access to a dashboard (Figure 3) with the following: an admin panel displaying the consent status of each user; the protocol that transparently stores the public keys of each consenting user (through Chainscript); and the history of each released version of the protocol and the consent and re-consent of the user attached to each of these versions.\n\nFor each user a pair of private-public keys were provided, (https://msdn.microsoft.com/fr-fr/library/windows/desktop/aa387460(v=vs.85).aspx). These are asymmetric cryptographic data that enables authentication on Blockchain. These were randomly attached in one-to-one correspondence to a user’s emails. We focused our interest to Blockchain’s usage in the time-stamping and archiving logic. We did not let users create or use their own Blockchain authentication setup, i.e. if a user owned a Bitcoin account, the key and Bitcoin address were not allowed to be used. This restriction was not related to Blockchain complexity, but rather to maintain a simple and common email-focused authentication process. Other ways exist for authentication, such as physical devices, like USB keys or cell phone fingerprints, but this would have led us far from the focus point of our protocol-related problematics.\n\nThe process that subjects were submitted is as follows:\n\n• The study investigators to send email to the patients.\n\n• The users receive the email, which contains a web hyperlink redirecting them to the web interface that displays the consent form.\n\n• In the background, after clicking the consent button without the user experience being bothered by any blockchain transaction related complexity, the user signature is registered to the Blockchain and is timestamped.\n\n• Each time the protocol is updated, investigators send an email explaining the major changes that occurred and users are invited to sign the revised consent form.\n\n• Each step of this process is updated on the investigators’ admin panel with the version of the consent document and the user's consenting status.\n\nSignatures of the evolving consent document were registered to the Blockchain. Moreover, all of the relevant interactions of the user with our platform was stored in the Blockchain, i.e. the consent form upload by the investigator; email requests to users; and patient signatures.\n\nThe piece of data attesting and synthesising all this information is called a Chainscript. It is a JSON formatted data structure holding all the information related to the protocol and users’ consent. Chainscript is a JSON format developed by Stratumn SAS, especially dedicated to attest the steps in trusted workflows (http://chainscript.io/). Chainscript is an open standard. The philosophy behind it is to be able to prove the integrity of a process with a single JSON data structure by securing the who, when, what and where of a sequences of steps that are linked together in chronological order. Each sequence corresponds to a segment, and each segment holds some metadata, an identifier called a hash, and a pointer to the preceding segment. The critical information maintained in Chainscript are the hashes, which are the proofs of the existence of data. Since each Blockchain transaction has a cost, we decided to group the transactions. What makes Chainscript interesting is that a series of Blockchain transactions can be wrapped into the same logic flow, preventing too intensive requesting to the Blockchain network, which can prove to be costly.\n\nTherefore, with this information, how can we check proof of a specific data after they are merged? The ChainScript solution relies on a singular data structure, a Merkle Tree, which in a way “hashes the hash”, preserving in one single hash all the hashes, so that if any hash is corrupted, the entire tree is invalidated.\n\nIn our implementation, the ChainScript code is held in the PDF consent document, storing its hashed content, all its versions (corresponding to protocol revisions) and all the signing users for each version. It is of major interest that ChainScript can be publicly verified without any proprietary tool.\n\nWe should remark that a positive side effect of tracking each step of a user’s interaction with the platform is that storing so exhaustively the data, raises the barrier to fraud.\n\n\nResults\n\nWe were able to collect consent and re-consent of users and store them in a transparent, unfalsifiable, verifiable way. These data were encoded in a single document. This document holds the stakeholders’ identifiers, the users’ identifiers, timestamps of the protocols being sent, consent status, timestamps of the consent, and the version of the protocol to which the consent is attached.\n\nThis master document was shared in real-time through key actors that ruled the POC study. It was registered to the Blockchain in a safe way, so that this group of stakeholders retains the timestamped proof of the consent status in an immutable document. We stress again the fact that this document is incorruptible, and it is possible to check its consistency on any dedicated public website (e.g. a website that enables checking of Bitcoin transactions), proving the correspondence between each version of the protocol and its related consent.\n\nTechnically, these data are synthesized in the open data format we mentioned earlier, Chainscript, which is as follows:\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\nAll this information is bound together in one data structure, so that the whole set of obtained consent, with their uniquely attached version of the protocol, form an immutable global data. The change of a single element breaks the entire data structure.\n\nIn the interest of user confidentiality, the master document cannot be made available.\n\nWe sent two versions of the protocol (Supplementary File 1 and Supplementary File 2) during the study, through which we sought the users consent; each consent was attached to a specific version of the protocol.\n\nUsers were given a digital signature and secured key, each of which consisted of a hash. Amongst the users of this experimental study, 14 gave their consent to the two versions of the protocol, 9 gave their consent to only one version of the protocol and 2 didn’t give their consents at all and 2 did not respond to any consent form.\n\nThe interaction of the user with the online interface, namely accepting or refusing to give consent, led to a transaction validated in Blockchain. Each version of the protocol had a unique identifier, called a hash. The hash is uniquely attached to the content of the protocol document. The correspondence between the consent document and the hash is one-to-one, namely if one single letter is changed then the hash is broken.\n\nFigure 4 shows where the identifier of the protocol document is highlighted in the Chainscript master document. Figure 5 displays the investigator identifiers, and Figure 6 reveals the consent status bound to the protocol revision version.\n\n\nDiscussion\n\nBlockchain is an emerging, fast-evolving technology. The boiling atmosphere around development and use of Blockchain is similar to tech development during the early stages of the web : It took several years before formatting as html or css became web standards. Blockchain technologies are gaining more and more attention, and Blockchain networks are proliferating, for example Ethereum, Bitcoin, Hyperledger or private Blockchain networks. It is not clear which of these will impose itself as a blockchain network standard, or even if there will be unique standard one. Public networks are interesting due to the idea of a community driven approach and scalability, but private ones can offer a certain level of customization.\n\nAlongside this fast-developing tech, there are still some infrastructure obstructions that are not yet circumvented, namely delays in transaction validation. On the Bitcoin network, the validation process (via the so-called mining) takes around 10 minutes (https://www.quandl.com/data/BCHAIN/ATRCT-Bitcoin-Average-Transaction-Confirmation-Time). In the present study’s context, we are not tied by real-time requirements measured in seconds, and so it is not a major obstruction. Moreover, the ChainScript logic we implemented in our POC allows grouped network request validation, which preserves the Blockchain network from computation overload, and allows to scale our method to a large patient cohort. More generally, to tackle this challenge of scaling the network, in case there is a massive amount of transactions, there are some implementations of Bitcoin-based protocol isolated from the Blockchain, the most renown is called SideChain (https://www.deepdotweb.com/2014/06/26/sidechains-blockchain-2-0/; https://www.reddit.com/r/Bitcoin/comments/2kdxy8/).\n\nMoreover, with regard to the authentication process, we can expect that, when the use of Blockchain’s technologies will be more common, there is an important chance that users already possess a Blockchain public-private-key identity. Therefore, sending keys for access and identification later in the signing process will be obsolete. This will require maintenance of a double key attribution (as explained above in the “Authentication method\" section) for users that do not have any Blockchain network identity and to be able to take into account those who have already one. In the latter case, verification of the digital signature of these users will have to occur.\n\nIt should be noted that we did not implement a consent revocation workflow. However, there is no issue in transposing at that end the Blockchain transaction logic we implemented for the consent. On that point, we should be careful about the fact that if the consent or its revocation can be given or withdrawn with no problem, these actions cannot be erased from the Blockchain. Indeed, if subjects revoked their consent by accident, then the action can be reversed, but data will remain containing the revocation of the consent and the cancellation of this revocation.\n\nFinally, we evoked a possible improvement in the enrolment rate, by empowering patients and granting them information and control over the enrolment phase. However, Blockchain is certainly not a “one size fits all” solution to the problem of a low enrolment rate. Indeed, there are many other parameters that interfere with the enrolment, which fall beyond the scope of transparency, user control and reliability that Blockchain technology helps to achieve: age, sex, cultural background, socio-economic factors, lack of educational materials16, readability and length of consent17–19, limited awareness about clinical research or patient-physician relationship20 and momentum of consent request21,22. Besides, the scope of our method did not address the question of consent collected in singular situations, such as intensive care, unconscious patients or psychiatric pathologies.\n\n\nConclusion\n\nKeeping track of consent collection is consolidated through the use of Blockchain technology. We have seen in this proof-of-concept study that all consent-related data can leave an unfalsifiable and verifiable fingerprint on the Blockchain. This is important both on the stakeholder’s side, letting them prove the existence and the consistency of the data, and on the patient’s side, giving them more visibility, transparency, and hence control over their consent.\n\nMoreover, though it was not be the focus of this paper, we anticipate that Blockchain technology, in that it does not rely trust on third party but inversely empowers peer-to-peer users by granting them control over consent agreement and revocation, can help gathering conditions of an improved privacy-respected freely-given consent. Besides, given its decentralized protocol, it can help introduce communities to contemporary clinical research, opening, for clinical reasearch, the path to implementing community management techniques in order to enrol patients using a more targeted approach.\n\nFrom a global perspective, the application of Blockchain technologies in the context of clinical research is broad and promising. Indeed, tracking the complex data flow with numerous diverse stakeholders, and documenting it in real-time through a timestamping workflow, is a key step towards proving data consistency and inviolability, and will hence improve clinical trial methodology.\n\n\nSoftware availability\n\nLatest source code available at: https://github.com/benchoufi/DocChain\n\nArchived source code as at time of publication: doi, 10.5281/zenodo.237040 (https://zenodo.org/record/237040#.WHSxorYrI_V)\n\nLicence: 3-clause BSD licence", "appendix": "Author contributions\n\n\n\nMehdi Benchoufi designed the work, initiated and led the analysis on blockchain impacts in the context of clinical trials. With the co-authors, he contributed to identify consent collection process as a substantial use-case for a blockchain implementation, in the idea of clinical trial transparency and traceability. He designed the blockchain collection website and was the medical coordinator of technical developments.\n\nRaphaël Porcher contributed to the design of the work, especially on identifying with the corresponding author which of the consent process steps could be wrapped into a Blockchain process. He has put in perspective the potential of implementing Blockchain in consent process, with regards to information of patients, ethics, data privacy.\n\nPhilippe Ravaud brought his expertise to consolidate the design of work, identified with the corresponding author the issues related to consent process that should be tracked through Blockchain transactions, especially the ones related to protocol revisions and lack of consent collection renewal. He analysed the results with Raphaël Porcher and the corresponding author and inferred from them improved methodology perspectives that Blokchain draw for the entire field of clinical research.\n\nBoth Raphaël Porcher and Philippe Ravaud reviewed the article, finally approved the article and agree to be accountable on any part of the article relatively of its accuracy and its integrity.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: Protocol and consent form (versions 0 and 1) used in the proof-of-concept study (in zipped file) (in French).\n\nClick here to access the data.\n\nSupplementary File 2: Protocol and consent form (versions 0 and 1) used in the proof-of-concept study (in zipped file) (in English).\n\nClick here to access the data.\n\n\nReferences\n\nGupta UC: Informed consent in clinical research: Revisiting few concepts and areas. Perspect Clin Res. 2013; 4(1): 26–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLloyd Wendy: BA, LPN, CIP,CCRP, Regulatory Affairs and Compliance Specialist Part 2 of 3 Part Series: Informed Consent, the process. Reference Source\n\nOffice of Scientific Investigations, Metrics. US Food and Drug Administration. 2014. Reference Source\n\nBarney JR, Antisdel M: Common problems in informed consent. Human Research Protection Program (HRPP). 2013. Reference Source\n\nSeife C: Research misconduct identified by the US Food and Drug Administration: out of sight, out of mind, out of the peer-reviewed literature. JAMA Intern Med. 2015; 175(4): 567–77. PubMed Abstract \| Publisher Full Text\n\nDillner L: BSE linked to new variant of CJD in humans. BMJ. 1996; 312(7034): 795–800. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWMA Declaration of Helsinki - Ethical Principles for Medical Research Involving Human Subjects. Reference Source\n\nMyles PS, Williamson E, Oakley J, et al.: Ethical and scientific considerations for patient enrollment into concurrent clinical trials. Trials. 2014; 15: 470. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInformed Consent Information Sheet Guidance for IRBs, Clinical Investigators, and Sponsors. Reference Source\n\nGupta UC: Informed consent in clinical research: Revisiting few concepts and areas. Perspect Clin Res. 2013; 4(1): 26–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCaldwell PH, Hamilton S, Tan A, et al.: Strategies for increasing recruitment to randomised controlled trials: systematic review. PLoS Med. 2010; 7(11): e1000368. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nResnik DB: Re-consenting human subjects: Ethical, legal and practical issues. J Med Ethics. 2009; 35(11): 656–657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcDonald AM, Knight RC, Campbell MK, et al.: What influences recruitment to randomised controlled trials? A review of trials funded by two UK funding agencies. Trials. 2006; 7: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSwanson GM, Ward AJ: Recruiting minorities into clinical trials: toward a participant-friendly system. J Natl Cancer Inst. 1995; 87(23): 1747–59. PubMed Abstract \| Publisher Full Text\n\nLovato LC, Hill K, Hertert S, et al.: Recruitment for controlled clinical trials: literature summary and annotated bibliography. Control Clin Trials. 1997; 18(4): 328–52. PubMed Abstract \| Publisher Full Text\n\nHazen RA, Eder M, Drotar D, et al.: A feasibility trial of a video intervention to improve informed consent for parents of children with leukemia. Pediatr Blood Cancer. 2010; 55(1): 113–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrehaut JC, Carroll K, Elwyn G, et al.: Informed consent documents do not encourage good-quality decision making. J Clin Epidemiol. 2012; 65(7): 708–724. PubMed Abstract \| Publisher Full Text\n\nPandiya A: Readability and comprehensibility of informed consent forms for clinical trials. Perspect Clin Res. 2010; 1(3): 98–100. PubMed Abstract \| Free Full Text\n\nParis A, Brandt C, Cornu C, et al.: Informed consent document improvement does not increase patients' comprehension in biomedical research. Br J Clin Pharmacol. 2010; 69(3): 231–237. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMills EJ, Seely D, Rachlis B, et al.: Barriers to participation in clinical trials of cancer: a meta-analysis and systematic review of patient-reported factors. Lancet Oncol. 2006; 7(2): 141–148. PubMed Abstract \| Publisher Full Text\n\nEder ML, Yamokoski AD, Wittmann PW, et al.: Improving informed consent: suggestions from parents of children with leukemia. Pediatrics. 2007; 119(4): e849–59. PubMed Abstract \| Publisher Full Text\n\nSmyth RM, Jacoby A, Elbourne D: Deciding to join a perinatal randomised controlled trial: experiences and views of pregnant women enroled in the Magpie Trial. Midwifery. 2012; 28(4): E478–85. PubMed Abstract \| Publisher Full Text" }	[ { "id": "19560", "date": "04 Apr 2017", "name": "Mike Clarke", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an important article, worthy of publication in F1000Research. There are some places in the article where the writing could be tidied up (e.g. references 1 and 10 are the same) but my main comments relate to questions that prompted in my mind, which the authors might wish to address if they revise it:\nProof of concept, blockchain and the study generally\nIs the reported study a \"proof of concept\" for the use in a real trial, or simply a demonstration that blockchain can be used for a series of sequential \"signings\"? If the latter, had that not been shown previously?\n\nAre there any plans to test this in a real trial, perhaps as a SWAT[1] and to include it in Trial Forge[2]?\n\nHow would this system be used if patients cannot get online personally?\n\nHow would the system cope if someone's email addresses changes? (I raise this because I am currently locked out of my Twitter account because the email I used to set it up is no longer active following my move away from that institution.)\n\nCan you reflect more on the challenges of doing online trials?\n\nDo you believe that this system will be applicable to all trials, a majority or a minority? You seem very enthusiastic about the use of this system and the article might benefit from the addition of more caution about its general applicability. For example, you write \"we evoked a possible improvement in the enrolment rate, by empowering patients and granting them information and control over the enrolment phase.\" but say little of the possible negatives (such as concerns about security of the data (see below); fear or discomfort with technology; and whether empowerment might come more from the ability to talk to a human being about the trial and the consent process).\n\nWho will ensure that blockchain is future proof? Might people need to print or export a copy of the electronic record for long-term storage?\n\nDoes blockchain allow for \"workarounds\" (e.g. to move to the next step without completing the previous one if for some reason this is necessary)?\n\nWhat do you mean by \"transparency\" in relation to the new system? If a potential participant thinks this means that others can see that they gave their consent, might this discourage them from joining the study?\n\nConsent in general\n\nHow would this system cope with differences between the process for obtaining consent to take part in prospective research in different countries and cultures? For example, what if someone other than the patient might need to give consent?\n\nWould patients be able to request that their ongoing consent is presumed without needing to be contacted again when there is a change in the trial? It might discourage patients from joining a trial if they are told that they will have to be contacted each time there is a change (especially if that change does not affect them personally).\n\nWhat protocol changes should lead to new consent (e.g. should it only be those that directly affect the patient, or should it be those that might have influenced their decision to join?)\n\nWould a patient need to be asked for their renewed consent if the change can no longer affect them? For example, if they have already completed treatment and are now on follow-up, do they need to be informed about changes in the evidence base about a side effect if they can no longer suffer that side effect?\n\nShould patients be asked to consent again, or be asked if they want to withdraw? What assumption would be made if they do not reply?\n\nMight it be worth discussing this new system in the context of other research into recruitment and retention (for example, as brought together in Cochrane Reviews [3,4,5,6]).\n\nIs a lack of informed consent a source of bias (or might it be closer to the \"truth\" if patients don't realise that they are being studied) or bad ethical practice?\n\nMight it be worth discussing the double standards of needing written consent for someone to receive a treatment in a trial but not needing it if they are given the treatment as part of \"routine practice\"?\n\nHow important is \"written consent\"? Is this unfair on difficult to reach populations who struggle to read or write?\n\nElectronic consent\n\nHow would you ensure that the appropriate person \"signed\" the consent form if you do not see them do so? Is it easier to submit someone's electronic key, than to forge their signature?\n\nSecurity\n\nMight patients' concerns over the security of their data and the importance of confidentiality make them cautious about joining a trial if they had to use this system? How worried might they be because of news stories about data from banks and other supposedly secure systems being hacked and leaked?\n\nMight it be worth writing something about how patients may think that paper consent forms locked in a filing cabinet are more difficult to access and make available to everyone online, than documents that are already available to the research team from anywhere on the internet.\n\nLanguage\n\nThe words \"subject\" and \"participant\" are used to refer to people who take part in trials. I prefer to avoid \"subject\".", "responses": [ { "c_id": "2656", "date": "27 Apr 2017", "name": "mehdi benchoufi", "role": "Author Response", "response": "Proof of concept, blockchain and the study generally Dear reviewer, Here are some responses to the questions that were raised. Besides, thank you for your remark related to the bibliography and the reference duplicate. 1. Is the reported study a \"proof of concept\" for the use in a real trial, or simply a demonstration that blockchain can be used for a series of sequential \"signings\"? If the latter, had that not been shown previously? In the idea of establishing all the consent process in real conditions, we claim it is a proof of concept. We developed a complete and realistic set of interactions between fake patients and stakeholders, and we paired consent status and protocol revision through blockchain held by a single master document accounting for the whole process. 2. Are there any plans to test this in a real trial, perhaps as a SWAT[1] and to include it in Trial Forge[2]? For the moment, we have no specific plan to implement in a real trial but our POC is precisely a preparation to go further to a real setting. We have no expertise in SWAT or in using Trail Forge platform, but it should comply with no difficulty with our implementation. 3. How would this system be used if patients cannot get online personally? This is more a problem related to online consent than a blockchain related question. In situations where patients cannot get online personally, then the medical doctor should provide the consent form to the subjects by hand. Besides, let's indicate that in most countries, the written consent is legally mandatory. There are other situations where the online access is not possible, related for example to disabilities, then either the legal representative should be given access to the online consent form, or written consent should be sought. 4. How would the system cope if someone's email addresses changes? (I raise this because I am currently locked out of my Twitter account because the email I used to set it up is no longer active following my move away from that institution.) In a real clinical trial, we would set multiple ways to reach a patient : digitally, phone number, postal mail. For sure, email is not sufficient, and we would use a stronger identifying system than email, i.e. material objects storing cryptographic keys, such as a USB key. We could also build a Smart Contract triggered by a destination email error callback. Then, when this condition is met, Smart Contract would cause the other reaching methods to be proceeded. Since Smart Contract are pieces of code, this automatized processed can be customized at will. However, although this is not the situation you are pointing to, this procedure may find a limit: there is no way to distinguish between the situation where an email has been sent without response, and the one where the email is still maintained by the institution with no access anymore for the previous account holder. But, we think this very case looks quite exceptional. 5. Can you reflect more on the challenges of doing online trials? There are some challenges regarding online trials: - people that are not in a condition to access an online platform: severe conditions, lack of consciousness, learning disability, no access to internet or people not friendly to online tools - the current state of technologies do not allow interventional trials. However, the explosion of IoT, miniaturization could lead to imagine some specific interventional trials to be conducted online. In this respect, we mention the existence of blockchain systems specifically dedicated to connected objects. - ensuring that the person consenting is effectively the one pretending to be: this should lead to use a strong identifier, and at minimum use KYC process (Know Your Customer) implemented to bind digital identities to physical entities in the case of sensitive data. Fiscal administration, Banks makes use of that. It is worth noting that from a blockchain point of view, the email is by no mean a method to identify. In our settings, we generate identifiers for each patient which consists in complex cryptographic strings. It would be possible in addition of these informations stored in the local machine of the subject, to duplicate this information on a USB key or identifier cards on the model of KYC procedure. Let's state that there are some companies providing material supports to keep the blockchain id's. 6. Do you believe that this system will be applicable to all trials, a majority or a minority? You seem very enthusiastic about the use of this system and the article might benefit from the addition of more caution about its general applicability. For example, you write \"we evoked a possible improvement in the enrolment rate, by empowering patients and granting them information and control over the enrolment phase.\" but say little of the possible negatives (such as concerns about security of the data (see below); fear or discomfort with technology; and whether empowerment might come more from the ability to talk to a human being about the trial and the consent process). Indeed, blockchain is not without carrying some issues. Our point was to put in a perspective a trend. Every new technological gap raises some fears, as the internet at its very beginning, but the overall trend is that usages have imposed. It is worth noting that users shape also technologies and their fear pushes the improvement of technologies conducting these to take more account of their needs and apprehensions. Besides, our idea is not to rely on a technology by itself but to complete the blockchain network ability to ensure data consistency by a strong human support at any step of the clinical trial, first and foremost the consent process. Besides, from the data security point of view, we positively think that it is much better ensured by blockchain-like technologies than the one currently used : first, because of the strong crypto-oriented transaction validation, second the distributed nature of the network prevent from the \"single point of failure\" problem related to centralized data collection. By the way, we can take the Bitcoin network as an example, which carries sensitive money data and which is proving to be resistant for almost ten years. 7. Who will ensure that blockchain is future proof? Might people need to print or export a copy of the electronic record for long-term storage? Blockchain consist of a network of peers, anybody involved in the network storing in his computer the archive of all the history of transactions, i.e. the public ledger. So that, even if the network had to fail for one or other reason, any single node can restore the last state of the network. Moreover, if necessary, depending on the design of the blockchain implementation, we might enable the only stakeholders or if we want any participant, to export and print the copy of electronic transactions. We may have in mind that data stored are \"proof of data\" that can be checked to match the true data on any dedicated public website. 8. Does blockchain allow for \"workarounds\" (e.g. to move to the next step without completing the previous one if for some reason this is necessary)? Yes, one core functionality of blockchain technologies, is Smart Contract. They allow to write algorithmically any set of conditions that modules the execution of some instructions. But, we stress the fact the system can be \"fault-tolerant\" but there are no \"roll-back\" possibility. So, suppose that someone stores some informations but has mistaken, then he'll be able to add the new corrected information (that's what we can call a \"fork\"), but the first errored information is still accessible in the blockchain. 9. What do you mean by \"transparency\" in relation to the new system? If a potential participant thinks this means that others can see that they gave their consent, might this discourage them from joining the study? We mean by transparency that the rules are clear for anybody taking part in the process. This is the very role of the Smart Contract we already mentioned. Besides, the perimeter of the people sharing the data can be controlled : we can decide that data can be shared by the only stakeholders, or decide that participants will be able to share access to their data with persons of trust. All this fine-grained control can be powered on the top of blockchain. Consent in general 10. How would this system cope with differences between the process for obtaining consent to take part in prospective research in different countries and cultures? For example, what if someone other than the patient might need to give consent? Differences between cultures, countries need to be tackled one by one. So that we need true implementation of clinical trials to face with those kinds of issues. I don’t think there is a one general answer. Online tools can help orienting the participant regarding to their specific situation. Moreover, we believe chat support should be provided in order to take account of specific situations 11. Would patients be able to request that their ongoing consent is presumed without needing to be contacted again when there is a change in the trial? It might discourage patients from joining a trial if they are told that they will have to be contacted each time there is a change (especially if that change does not affect them personally). Indeed, there is no need to overwhelm patients with a flood of informations. However, from a general point of view, patients association often complain about lack of informations regarding the clinical trial progress, so that some calibrated informations can be delivered conveniently to the patients. Moreover, in case of a major change in the protocol, they should be specifically targeted to get an email asking to consent again. 12. What protocol changes should lead to new consent (e.g. should it only be those that directly affect the patient, or should it be those that might have influenced their decision to join?) There is heavy litterature about this. We refer it in the article: [10,11,12] in our bibliograhy and we mentionned these links detailing the protocol changes that should lead to renew the consent: http://www.irb.pitt.edu/sites/default/files/reconsent guidance.pdf; http://www.mayo.edu/research/documents/29-re-consent-or-notification-of-significant-new-findingspdf/doc-10027714; http://www.yale.edu/hrpp/policies/documents/Reconsentingguidance.pdf) 13. Would a patient need to be asked for their renewed consent if the change can no longer affect them? For example, if they have already completed treatment and are now on follow-up, do they need to be informed about changes in the evidence base about a side effect if they can no longer suffer that side effect? Yes, these kind of new informations should be given to patients and require to ask for a renewed consent again, even if they are not supposed to suffer this side effect thereafter. 14. Should patients be asked to consent again, or be asked if they want to withdraw? What assumption would be made if they do not reply? If patients want to withdraw, they should not be asked again. When patients do not reply, and after ensuring by other means (emails, mail, phone call if available) that they do not provide an answer, then they should be considered has consenting because they already gave their consent. Besides, this kind of situation can be advantageously scheduled in a Blockchain Smart Contract, that can be adapted to local legal contexts. 15. Might it be worth discussing this new system in the context of other research into recruitment and retention (for example, as brought together in Cochrane Reviews [3,4,5,6]). Indeed, we might implement some Smart Contracts, checking the recruitment and retention process. So Cochrane reviewers could have an insight about whether this process was done conformally to standard procedures. However, we notice that the code of the Smart Contract should also be reviewed, so that an experienced developer should be required. 16. Is a lack of informed consent a source of bias (or might it be closer to the \"truth\" if patients don't realise that they are being studied) or bad ethical practice? Lack of informed consent is for sure a bad ethical practice, in a strict contradiction to Helsinki declaration, Nuremberg declaration and good clinical practices. Regarding the matter of generalisability bias, the latter is more related to the setting of inclusive criteriae than related to consent. 17. Might it be worth discussing the double standards of needing written consent for someone to receive a treatment in a trial but not needing it if they are given the treatment as part of \"routine practice\"? In any case, the participant to a clinical trial must to sign the consent form, even he is already taking the medication for which the consent form is seeking his/her consent. At this occasion, the patients may be informed of actual informations related to the treatment, for example new side effects of a drug. 18. How important is \"written consent\"? Is this unfair on difficult to reach populations who struggle to read or write? The written status of the consent by opposition of the electronic has not by itself more credit. However, depending on the local legal context, “written consent” is mandatory. Besides, collecting consent of people with reading, writing or learning disabilities needs care. The person collecting the consent must assess the ability of the person to understand correctly the informations and to make a decision. The information must be given orally and to the legal representative if any. Some ethics committees allow the mediation of families or other supports at this stage. Electronic consent 19. How would you ensure that the appropriate person \"signed\" the consent form if you do not see them do so? Is it easier to submit someone's electronic key, than to forge their signature? In this POC, we generated cryptographic keys for each patient. So, in this design indeed, we can't ensure that the person consenting is the person he or she pretends to be. This can be improved in different manner. At least, using KYC standard procedures, i.e. doubling the electronic identification by one related to a physical object holding some number code. One step further would be to provide patients with an objects storing USB key storing the cryptographic signature and unlocked by a easy-to-remember id. Security 20. Might patients' concerns over the security of their data and the importance of confidentiality make them cautious about joining a trial if they had to use this system? How worried might they be because of news stories about data from banks and other supposedly secure systems being hacked and leaked? We refer to the question 6 on the notion of “single point of failure”, which answers at some level the raised issue. In any case, patients are very sensitive to the security and the privacy of their data and this system addresses precisely this issue. Indeed, the decentralized structure of the blockchain, the involvement of anyone as a peer on the network, the ability to finegrain the data sharing perimeter, allows more control of the patients over the data workflow. However, we are conscious that this system is very new and needs some pedagogical support. Anyway, Bitcoin is now a widespread, trusted electronic currency, available on payment platform of a wide range of websites such as Amazon, Apple’s App Store. An implementation of such processes in a real clinical trial should be the occasion to add different media support : documents, videos in order to inform patient of the benefit of using such technologies . For the second part of the question, see the response to question 6. of the present reviewing question list. 21. Might it be worth writing something about how patients may think that paper consent forms locked in a filing cabinet are more difficult to access and make available to everyone online, than documents that are already available to the research team from anywhere on the internet. Absolutely it might be very interesting. Language 22. The words \"subject\" and \"participant\" are used to refer to people who take part in trials. I prefer to avoid \"subject\". We substituted the usage of \"subjects\" by \"participants\" in the revised document. Best regards," } ] }, { "id": "21311", "date": "11 Apr 2017", "name": "Daniel S. Himmelstein", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBenchoufi et al. propose and implement a method for notarizing participant consent for clinical trials using the Bitcoin blockchain. At a minimum, such an approach must accomplish two cryptographic objectives:\nprovide participants with a fraud-resistant method to irrefutably consent to the terms of a clinical trial. provide clinical trial investigators with a method to retrospectively verify participant consent at a given point in time.\nI agree with the authors that cryptographically notarizing consent would be a major advance. If possible, there would be strong incentives, both ethical and practical, for investigators to implement and regulatory agencies to mandate such an approach.\nBy encoding a hash into the Bitcoin blockchain that derives from a \"consent document\", it is possible for investigators to timestamp a consent document and thereby retrospectively prove the existence of that consent document at a given point in time. However, I am not convinced that the study provides patients with a fraud-resistant, irrefutable method of consent. Specifically, I don't think the study provides a method for ensuring that a specific participant provided consent. In other words, can clinical trial investigators prove that a specific participant consented rather than just proving that some entity consented under the supposed digital identify corresponding to a specific participant?\nThe study uses digital signatures to attest that a participant approved a specific consent form. The implementation relies on bitcoin private keys to provide digital signatures. The problem is that bitcoin addresses (which uniquely derive from a private key) are pseudonymous. For example, anyone with access to a consent form could create an unlimited number of bitcoin private keys and use these private keys to digitally sign the consent form. How does one prove that a bitcoin address solely belonged to a single participant?\nThe generation of bitcoin private keys in this study occurs at https://git.io/vSPTE. I don't see anything in the code or manuscript that irrefutably links a participant to ownership of a given bitcoin address. Therefore, it appears to me that clinical trial investigators (or many other actors) could forge consent. In fact, the manuscript appears to concede this point with the statement:\n> Each of the to-be-enrolled users were assigned a private key in order to sign data and documents, and in practice this would be used to publish their signed consent.\nLinking digital identities to physical identities is difficult. However, this is a precondition to blockchain notarization of clinical trial consent. OpenPGP (the most established method for digital identity and signatures) relies on a web of trust model to link digital identities to physical identities. HTTPS relies on Certificate Authorities to link digital identities to organization identities.\nSince the manuscript does not sufficiently address how it links digital and physical identities, I dug a bit deeper into Stratumn, the underlying service provider. Stratumn is a French company, which aims to \"secure processes between partners through blockchain technology.\" Stratumn focuses on \"proof of process\" using a JSON data structure called chainscript. I had difficulty uncovering the implementation details of Stratumn's proof of process, as much of the available online material focuses on the implications of the technology rather than the technology itself. The most helpful resource I found was the Epicenter Podcast #159 titled \"Richard Caetano & Anuj Das Gupta: How Stratumn Secures Processes.\" This investigation did not answer how digital identities are linked to physical identities in Stratumn's services. The Stratumn proof-of-process white paper does mention identity verification under \"Non-Repudiation of Source and Destination\", stating:\n> Non-repudiation implies that the stakeholders of the information content of each and every step should not be able to deny their involvement with the steps representing their data through the digests. The tool we will use for this is Digital Signatures.\n> Both Alice and Bob need to be responsible to their respective steps in such a way they they can not repudiate their involvement if challenged. The record of their identities would be maintained by having the stakeholders digitally sign the digest of their move and then storing the signatures and public keys along with the digest in the step. The private keys will not be stored in the steps; each player hold hers separately and securely. Anyone who has access to the proof can use the public key verification to ascertain whether or not Alice or Bob can be held responsible for a step.\n> In this way we enable identity management and ownership in each and every step for the proof of a process to demonstrate the Who behind each and every step.\nUnfortunately, this description does not explain how digital identities are linked to physical identities. How does one know whether a digital signature is actually Alice or Bob's? Stratumn even provides a document detailing the clinical trial consent use case. However, this document does not provide an identity solution.\nConclusion\nI am marking my review as Not Approved. If the authors can show that it is not trivial to forge a specific participant's consent, I would be happy to revisit my decision. However, absent a reliable method to link a digital identity to a participant's physical identity, there is little benefit to cryptographic notarization of clinical trial consent. Such an approach is only as useful as its most vulnerable step. At a minimum, the authors need to identify the trusted parties related to participant identity.\nMinor points\nThe study could do a better job citing the relevant cryptographic literature. For example, the study cites neither the Bitcoin white paper nor the proof-of-process white paper. In addition, the study should consider referencing OriginStamp, OpenTimestamps, and Carlisle's 2014 blog post.\nFigures 2 & 3 are in French. I understand that it's important to show the consent form and interface as given to the participants. However, perhaps these should be supplements with English versions in the main text.\nThe study states: \"Smart contract: this is a contract that is algorithmically implemented and binds any change in the protocol to the patients’ consent seeking renewal.\" However, from my understanding the study does not propose any blockchain smart contracts. Instead, the Bitcoin blockchain is only used as a timestamping service.\nPositives\nThe study aims to replace trust with cryptography in medical research.\nThe study makes its source code available under a permissive open source license on GitHub (Zenodo archive).\nThe study understands that directly writing every document hash to a secure & immutable blockchain will be cost prohibitive, and therefore it is necessary to \"group transactions\", i.e. write one transactions that attests to the existence of many chainscript hashes.", "responses": [ { "c_id": "2657", "date": "27 Apr 2017", "name": "mehdi benchoufi", "role": "Author Response", "response": "Dear reviewer, Thank you for drawing our attention on the questions you raised. Our answer focuses mainly on the issue related to the binding between digital identities and physical entities. We updated the revised version accordingly to all the questions you raised. Target of the POC and digitale identities POC as a response to FDA raised issues Thank you for your constructive remarks. In fact, there are two main issues regarding the consent process. The first one is related to the quality of the process itself and the second one is related to the identity of the individual consenting. Monitoring of trials by the FDA has identified serious issues in about 10% of trials, major concerns being failure to obtain written informed consent, consent document not signed or not dated, missing pages in consent document provided to subjects,failure to re-consent when new information becomes available, use of expired, non-validated or unapproved forms (reference [3,4] in the manuscript and this link). In this POC study, we aimed at fighting these issues, where existing patients were included in a study in the presence of their physician or staff. In our implementation, we ensured that all the consent process was tracked in time all along the inclusion period, that the documents made available to the subjects were not corrupted, that they were in conformity with the current version of the protocol, and that (re-)consent was asked as many time as required. We however did not address the cases where consents were falsified by the investigating teams or where fake patients were invented for instance, which are more related to the second (identity) issue. This should be more explicit in the text, and has been emphasized in the revised manuscript. Linking digital identities and physical entities We then agree with the reviewer that the second issue is important in the context of a real clinical trial. This relates to the question of pairing digital identification with physical persons. In our implementation of the POC study, the link between a participant and his/her digital identity is done through his/her email address. A participant cannot simply generate any Bitcoin address and use it to sign, because the private key is created on the server by the agent (but not saved on the server), then is sent to the email address. So the only guarantee is that the participant had access to that email address. This is pretty light, but seemed to be satisfactory given the scope and focus of the POC. Moreover, in the setting of a real online consent process, there is no chance that a patient who would not effectively consent—for instance if there were some fraudulous operation registering him/her as a consenting subject—would actually participate to the study. However, in a production application we must implement a more secure mechanism, and we thank the reviewer again for outlining the possibilities. There are indeed several solution to secure the digital identity of participants. First, we can mention that KYC processes are now quite widespread and there are plenty of examples where digital identities are linked to physical entities or human beings in the context of sensitive data: in most western countries the fiscal administration allows online service for taxpayers to declare their income and proceed to related operations, banking services also provides their services online such as bank transfer, account consultation. All these examples have in common that users (taxpayers, bank clients) are given a physical material, often by sending a document by regular postal service, or by giving a first card or document by hand. These documents carry an identification number. This approach could be implemented in production. Second, even in regular off-line consent process, ensuring that the patient really signed the document is not without carrying problems. We have in mind that the patient must meet the medical doctor and receive by hand their consent document. So that, when they go back to the medical doctor with the signed document, there is no way to prove that it was signed by the patients himself or that the patient was not influenced at the time of signing the consent form. Regarding a possible implementation of our blockchained consent process in a real clinical trial, we can provide, at the moment the patient meets the doctor, authentication cards with an identification number and so strengthen the binding between the electronic signature id and the physical one. Let’s mention that some bitcoin companies also provide material keys, such as USB keys, that hold the cryptographic signature, which can be unlocked by an easy-to-remember code. This can be an excellent candidate to get stronger digital-physical binding. These issues are now covered in the revised discussion. Besides, we think that full online consent collection process raises more the issue to reach subjects that the one related to ensuring the targeted patient is really the one consenting. Patients invention Going further, we can summarily refer to the extreme case of patients invention. Unfortunately, there is almost no way to prevent data invention. And there are some documented case in the medical literature, fortunately very rare, where clinical trial patients and all related clinical data were invented from end to end. Let’s notice here that, in the first implementation of our POC, we timestamped an extensive part of interactions between the subjects and the online email and web platform : time of the email sending, reception, time email was opened, the links being clicked on. We then validated the transactions into the blockchain, this way we could at least detect anomalies if such data appear grouped in time, so that it does not prevent from invention but higher the barrier to fraud. Further technical improvements On a more technical side, Stratumn’s technology, via proof-of-process, provide an implementation that would allow one to create an audit trail of the the different steps that happened until the participant provided enough evidence to convince the verifier of his/her identity, which is quite an upgrade to current KYC processes, because there would be a permanent record of what happened, and the process would impose rules that must be respected to the letter (by computer code) before moving to the next step. In short, this is not a complete out-of-the-box solution to linking digital identities, but Stratumn has the tools to build one. While implementing such a verification process with the technology is possible, this was beyond the scope of the POC. Smart Contracts Indeed, we did not implement it in our setting, we meant to mention that Smart Contract can bind the modifications occurred in the protocol and some to be defined events on which parties can agree. Besides, we did not detail or implement in the article all the advantages that can be derived from the algorithmic nature of the Smart Contracts: the way consent process is monitored, the way the related informations are shared between the stakeholders, the investigators and the IRB or building Smart contracts with the condition patients will only be included when the enrolment is complete or building one checking the whole consistency between the data and the blockchained proof of data, preventing from the whole hassle of gathering the documents and checking them by hand Figures Indeed, some figures are in french and we complied to the F1000 policy that invited us to proceed so. Bibliography We thank the reviewer for the precious remarks about bibliography and to have brought to the knowledge of the authors two of them, namely the one referred as `OriginStamp` and the Carlisle blogpost. We add and referenced them in the revised document. Best regards," } ] } ]	1	https://f1000research.com/articles/6-66
https://f1000research.com/articles/7-133/v1	01 Feb 18	{ "type": "Opinion Article", "title": "Hubris and Sciences", "authors": [ "Eleftherios P. Diamandis", "Nick Bouras", "Eleftherios P. Diamandis" ], "abstract": "There has been an increasing awareness of the importance of leadership and decision making, including scientists and academics, over recent times. By whom and how decisions are made can have serious implications across all levels of society. Several people have been successful in their life and have been inflicted by excessive pride and self-confidence. There are times when the manifestations of such behaviours demonstrate noticeable signs of narcissism and on extreme cases, hubris. Hubris is an old concept originated from the Greek mythology. The risk of hubris affects politicians, leaders in business, scientists, academia, the military, entertainers, athletes and doctors (among many others). Power, especially absolute and unchecked power, is intoxicating and is manifested behaviourally in a variety of ways, ranging from amplified cognitive functions to lack of inhibition, poor judgment, extreme narcissism, deviant behaviour, and even cruelty. Hubristic behaviour of overconfidence, extreme pride together with an unwillingness to disregard advice makes powerful people in leadership positions to over-reach themselves with negative consequences for themselves and others. As the dangerous consequences of hubristic behaviours become more apparent and well described it is imperative that individuals, organisations and governments act to prevent such phenomena. Responsible leaders, including acclaimed scientists should exercise greater humility to the complexity and inherent uncertainty of their activities and strive to seek out and challenge hubristic behaviours.", "keywords": [ "Leadership", "Hubris", "Behaviour", "Science" ], "content": "The concept\n\nThere will be times when we think that audacity is the route to major discoveries and scientific breakthroughs. How often, however, do we question ourselves if what we know is a real knowledge and not some kind of information? How long will it take for the knowledge that is currently known to become outdated or obsolete? “I learned early in my career the dangers of being too entrenched in what I knew” stated Elisabeth Nabel1, Professor of Medicine at Harvard Medical School, revealing “how limits of knowledge can be a weakness and how ignorance can be strength”. She continued by saying that “none of us in science and medicine have the answers we tell others we have, because the universe of what we don’t know dwarfs what we do know”.\n\nThere are times in our life that we must accept that what we thought to be correct will be redefined. For example, stomach ulcer was believed for decades to be related to stress and proved to be a bacterial infection. There are also times we would felt that our pride was hurt, or our confidence was cracked by facts that we had accepted as certain. There is nothing wrong about being wrong. Consequently, humility is essential to be embedded by all of us in everyday life. We should be ready to accept that most of what we know might change over time.\n\n\nHumility\n\nHumility is defined by the Oxford dictionary as the “quality of having a modest or low view of one’s importance”. Humility is the element that prevents a sense of overconfidence and exaggerated self-importance and helps to avoid being inflicted by hubris. The Greek philosopher Socrates was proclaiming that “one thing I know is that I do not know anything”, which epitomizes his sense of humility. It is rather surprising that humility is not formally taught at any level of education. Most of us are left to self-discover humility, and many, never discover it. The opposite may happen too. A number of objectively clever, creative and successful people may develop megalomania, which, when left unchecked, can reach the level of hubris.\n\nHumility is necessary in science and technology since the remarkable contemporary advances and discoveries could encourage a feeling of acquired excessive power, with the consequence of developing hubris. Remarkably, some recent discoveries, such as the CRISPR-Cas gene editing technology, may encourage some to “play God” due to their supposed capability to alter the natural abilities of future generations of humans through genomics. In such instances, the discoverers of such technologies forget that they did not really create anything themselves, but they merely understood how microbes developed these tools through millions of years of evolution. The huge financial and commercial pressures affecting scientists in current times, together with those in academia for increased productivity, successful tenders for research grants and high impact factor publications add up to vulnerability of exhibiting behaviour and personality changes or traits manifested as hubris. Many successful people have frequently overestimated their own abilities and believed that their performance is superior to others by imposing excessive pride and self-confidence to themselves. There are times when the manifestations of such behaviours demonstrate evident signs of narcissism and on extreme cases, hubris.\n\n\nHubristic behaviour\n\nThe term hubris derives from the Greek mythology, signifying the dangerous combination of over-confidence, over-ambition, arrogance and pride. In the ancient Greek world hubris was considered to be one of the most dangerous traits one could exhibit. In the classical Greek myth when Daedalus and Icarus escaped from the labyrinth in Crete, Daedalus advised his son not to fly too low, in order to avoid being too close to the moisture of the sea or, too high and close to the heat of the sun, as he had used thread and wax to make their wings. Despite this advice, an excessively exuberant Icarus flew too close to the sun and his wax wings melted causing him to fall into the sea. Icarus’ hubris, his disobedience of his father in flying too high, is a cautionary tale about humility and restraint, the danger of audacity.\n\nHubris has been seen in all walks of life including politics, business, the military, scientists, academia, entertainment, sports and medicine (among many others). In aviation, investigations into fatal plane accidents identified erroneous decisions by the captain whose position of power in the flight deck dismissed concerns by other members of the crew. Moreover the crew often failed to question or challenge the captain’s decisions2. In medicine Atul Gawande (2014)3 suggested that the behaviour of doctors medicalising old age and not accepting that life/death is not curable is a sign of hubris within the profession. He argues that doctors should shift away from simply fighting for longer life, for things that make life meaningful. A leading Medical School in UK decided that it is no longer enough to have high grades to become a medical student but would-be doctors must also display humility4.\n\nPhenomena of hubristic behavior were possibly present in investigators of at least some of the fraudster studies reported in recent years. Among numerous examples, three articles are cited here one from the physics world and two from medicine. Jan Hendrik Schön rose to prominence after a series of breakthroughs in semiconductors, most of them published in Nature and Science, which were later discovered to be fraudulent5. Hwang-Woo-suk, until 2005, was considered one of the pioneering experts in the field of stem cells and was best known for two articles published in the journal Science in 2004 and 2005, where he reported that he had succeeded in creating human embryonic stem cells by cloning6. He was called the \"Pride of Korea\" in South Korea. These reports were later found to be fabricated. Another tragic example of possible hubris was the report in Nature of a new and simple way to produce inducible stem cells. The method was soon found to be irreproducible and was retracted, but in the meantime, one of the senior authors committed suicide7\n\nThe “intellectual celebrity syndrome” was implied by Winkler (1987)8 for writers and scholars who risk seeking to popularise serious ideas or influence contemporary events by transmitting them to the general public in a distorted and unusable manner. Winkler suggested that this phenomenon resembles a disease characterised by the presence initially of a pleasant exhilaration, followed by celebration, eccentric indulgent and somnolent phantasies. Nobel Prize laureates who undertake projects or accept positions beyond their capabilities was described by Diamandis (2013)9 as “Nobelitis”. Diamandis claims that the Nobel Prize seems to provide laureates with reassurance that they hold some super-powers that they did not realise before and that the prize will assist them to go on to even greater achievements.\n\nThe speed of communications in contemporary times, combined with the widespread application of social media and easy access to large groups of people, might predispose to both collective and individual hubristic decision making. The consequences of hubristic behaviour can be profound with dangerous consequences10. Acclaimed scientists should exercise greater humility to the complexity and inherent uncertainty of their activities and strive to seek out and challenge hubristic behaviours. Mentors are encouraged to discuss hubris and related behaviours with their mentees and stress the importance of humility in their future activities.", "appendix": "Competing interests\n\n\n\nNick Bouras was a member of the Advisory Group of Daedalus Trust\r Eleftherios P. Diamandis holds a consultant/advisory role with Abbott Diagnostics\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nhttp://www.tedmed.com/talks/show?id=309146.\n\nHelmreich RL, Merritt AC, Wilhelm JA: The evolution of Crew Resource Management training in commercial aviation. Int J Aviat Psychol. 1999; 9(1): 19–32. PubMed Abstract \| Publisher Full Text\n\nGawande A: Being Mortal: Medicine and what matters in the end. Metropolitan Books, 2014. Reference Source\n\nhttp://www.telegraph.co.uk/education/educationnews/4793869/Medical-students-must-now-have-humility-as-well-as-straight-As.html.\n\nService RF: Scientific misconduct. Bell Labs fires star physicist found guilty of forging data. Science. 2002; 298(5591): 30–1. PubMed Abstract \| Publisher Full Text\n\nHwang WS, Ryu YJ, Park JH, et al.: Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science. 2004; 303(5664): 1669–74. PubMed Abstract \| Publisher Full Text\n\nAnonymous: STAP retracted. Nature. 2014; 511(7507): 5–6. PubMed Abstract \| Publisher Full Text\n\nWinkler JT: The intellectual celebrity syndrome. Lancet. 1987; 329(8530): 450. Publisher Full Text\n\nDiamandis EP: Nobelitis: a common disease among Nobel laureates? Clin Chem Lab Med. 2013; 51(8): 1573–4. PubMed Abstract \| Publisher Full Text\n\nGarrard P: The Leadership Hubris Epidemic: Biological Roots and Strategies for Prevention. Palgrave Macmillan, 2017. Publisher Full Text" }	[ { "id": "30450", "date": "16 Feb 2018", "name": "Eleni Palazidou", "expertise": [ "Reviewer Expertise Psychiatry – mood disorders (depression bipolar disorder)", "psychopharmacology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe ancient Greeks recognized the dangers of “overvaluing” one’s self and hence coined the word Hubris (Ύβρις), meaning a highly exaggerated sense of self-importance that is an insult to the Gods.\nAnybody in position of power, in any sphere of life and the authors comment on scientists being afflicted by hubris as much as others. Very rightly they point out that scientific knowledge continuously grows and reinvents itself. In fact we can confidently (but not hubristically!) say, scientists will never manage to know everything! The growth of knowledge in every sphere is infinite.\nThe authors comment on the “intellectual celebrity status” which has afflicted scientists. “Nobelitis” is mentioned as a syndrome of considering oneself as more able than one is, because of the high level of recognition afforded to them, which encourages hubristic behaviour. Is prize giving, in general, feeding into hubris? It would have been good to expand on this.\nThey recommend the cultivation of humility as a way forward and they urge mentors to teach the importance of humility and the dangers of hubristic behaviour. Can humility be taught and hubris prevented? The public’s thirst for celebrity status, which doesn’t spare the science world, works against this.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "30448", "date": "12 Mar 2018", "name": "Eugene Sadler-Smith", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI enjoyed reading this interesting, relevant and well written article. I think that's it's great strength is the focus on hubris in scientific discovery - an area that is currently not written much about. I liked the examples and the arguments and I think they are convincing. If there are any areas for development maybe the authors could say a bit more about one of the really interesting points that they mention but don't develop -that is teaching humility, this is a really great and novel idea in hubris research and I like the idea of approaching it from a Socratic or Aristotelian perspective. Good luck I hope this work gets indexed.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-133
https://f1000research.com/articles/6-1824/v1	11 Oct 17	{ "type": "Software Tool Article", "title": "CellMap visualizes protein-protein interactions and subcellular localization", "authors": [ "Christian Dallago", "Tatyana Goldberg", "Miguel Angel Andrade-Navarro", "Gregorio Alanis-Lobato", "Burkhard Rost", "Tatyana Goldberg", "Miguel Angel Andrade-Navarro", "Gregorio Alanis-Lobato", "Burkhard Rost" ], "abstract": "Many tools visualize protein-protein interaction (PPI) networks. The tool introduced here, CellMap, adds one crucial novelty by visualizing PPI networks in the context of subcellular localization, i.e. the location in the cell or cellular component in which a PPI happens. Users can upload images of cells and define areas of interest against which PPIs for selected proteins are displayed (by default on a cartoon of a cell). Annotations of localization are provided by the user or through our in-house database. The visualizer and server are written in JavaScript, making CellMap easy to customize and to extend by researchers and developers.", "keywords": [ "subcellular location", "biological visualization", "protein-protein interaction" ], "content": "Introduction\n\nMany tools visualize different aspects of protein-protein interaction (PPI) networks; the most prominent might be Cytoscape1. Existing visualizations of large PPI networks continue to be difficult to use. Some proteins interact with many hundreds or thousands of others. Often referred to as ‘PPI hairballs’, such hubs are in the way of understanding large data sets. Many ways have been proposed to resolve such hairballs through the addition of biologically meaningful dimensions such as pathways2 or time3.\n\nAnother dimension was first introduced a decade ago, namely the overlay of PPIs with subcellular localization4. Combining PPI networks with protein location provide an intuitive way of laying out PPI networks on a graphical representation of the cell, and might reduce the clutter from PPI hairballs. This decade-old solution4 no longer copes with today’s data, in terms of scalability nor of customizability and in terms of ease-of-use.\n\nCellMap, the prototype introduced here, takes up on the idea of PPI visualization constrained by protein location, and provides a simple visual interface for users to explore protein location inside a cell. It presents this information in a graphically pleasant way and offers several customization features. The framework has been optimized to simplify future developments, such as the addition of further data dimensions (e.g. inclusion of protein trafficking). An instance of the tool with localization data from 5 and PPI data from 6 is available at http://cell.dallago.us.\n\n\nMethods\n\nThe CellMap prototype is an integrated portal that exposes API calls to retrieve images (representing cells) and protein information, as well as a frontend to visualize protein location and PPI data. The portal is fully written in JavaScript, namely in the JavaScript interpreter node.js (https://nodejs.org) for the backend and vanilla JavaScript for the frontend. The portal is deployed to the public through a Docker container. Docker is a technology that allows shipping of packaged services such as web applications to customers and users without the need to install dependencies other than the Docker engine (available through: https://www.docker.com). For the representation of cell images as maps, the Leaflet framework is used. Leaflet is a JavaScript-based tool used to represent maps (http://leafletjs.com).\n\nData about proteins are stored as JSON documents in a Mongo (http://mongodb.com) database. All information about the interaction partners and the subcellular localization of a protein is stored in a single JSON document, making the data structure simple to understand for non-experts and enabling them to deploy prototypes using their own data. Figure 1 schematically represents a protein data model (for a specific example for a protein object: http://cell.dallago.us/api/proteins/search/Q99943).\n\nIn CellMap, users can choose to upload new maps (images of cells). They can modify the location of regions of interest (ROIs) for a selected map (Figure 2), and visualize the locations of selected proteins on a map or render protein-protein interaction networks from a set of selected proteins.\n\nTo maintain a consistent coloring scheme for different cellular compartments throughout a set of different images, each compartment is assigned a unique color through the hash of the compartment’s name (e.g. light blue = vacuole, Figure 3B). Using this coloring approach, users might eventually learn to associate color with compartment. When proteins are loaded into the map, they are assigned pseudo-random coordinates representing a point that lies within the boundaries of the ROI in which they are localized (Figure 3D). A circle of a given radius is placed on the randomly generated point (Figure 3E-F), and the circle will be filled with the same color as the compartment in which the protein is located in (Figure 3B and 3F).\n\nIn the figure we present a diagram of the Protein class, which contains several attributes of type String, two fields of type timestamp and two arrays (in square brackets) that reference the Interactions and Localizations classes. The arrows highlight the referenced models. This simple representation of information about a protein, its protein-protein interaction partners and its localizations enables the tool to be reused with one’s own datasets.\n\nIn the screenshot, an authorized user with editing capabilities draws a polygon (dark green) representing a new cellular compartment or region of interest (ROI). The user has a set of tools on the left side that can be used to draw polygons, lines, squares or circles. Once the new region has been drawn, the user can associate a cellular compartment through the dropdown input on the top-right and submit the new information to the server. The image used for this screenshot was taken from Wikimedia’s user Royroydeb, under CC BY-SA 4.0 (http://bit.ly/2fuYRiE) and is used in this figure for demonstrative purposes only, as using it on the online version of CellMap would infringe copyright.\n\n(A) Section of a cartoon image of a cell; (B) user-drawn polygon representing the area occupied by a vacuole; (C) how the section of the cartoon image is displayed on the PPI/map viewer; (D) random point calculation inside vacuole-polygon-defined area; (E) drawing of a protein circle located inside the vacuole, (F) result of loading a protein localized in the vacuole as shown by PPI/map viewer.\n\nUsers can choose between two visualization options: the subcellular location in the context of the protein-protein interaction viewer (PPI viewer, Figure 4A, http://cell.dallago.us/ppi), and the protein subcellular location viewer (Map viewer, Figure 4B, http://cell.dallago.us/map). The two viewers can load the same images of cells (maps) and collect localization data from the same source, in the publicly available instance by5. The PPI viewer offers the possibility to overlay networks between proteins being visualized. The map viewer displays all locations reported for a given protein simultaneously, while the PPI viewer only displays one location at a time; users can manually change the location by clicking on the protein circle and selecting a new location from the information box (Figure 5). Both the PPI and the map viewer are enriched by several controls (Figure 6): The top-left controls enable actions including: the navigation to the home of CellMap (Figure 6, panels 1 and 2, A), switching from the map viewer to the PPI viewer and vice versa, keeping the proteins currently loaded in the view (Figure 6, panels 1 and 2, B), reducing the opacity of the cell map, highlighting the protein circles (Figure 6, panels 1 and 2, C), zooming in- and out of the map and PPI viewers (Figure 6, panels 1 and 2, D), and visualization of the global network among all proteins loaded in the visualizer (Figure 6, panel 1, E). The top-right control allows to temporarily hide loaded proteins or activate an overlay of the user-drawn localizations (Figure 6, panel 4). The top-center search panel allows users to load new proteins by searching for their UniProt identifier, primary gene or primary protein name7 into the viewer (Figure 6, panel 3).\n\nThe left view (A) shows the PPI viewer, which depicts the result of loading protein Q9NR71 and displays a circle for the first localization found in the array of locations (http://cell.dallago.us/ppi?p=Q9NR71); The right panel (B) shows the Map viewer, which depicts the result of loading the same protein Q9NR71 and displays a circle for the protein in each of its reported location (http://cell.dallago.us/map?p=Q9NR71). The red arrows are overlaid on top of the screenshots to highlight where the protein circles have been drawn in the viewers, since fitting the screenshot on the page reduces the overall size of the images.\n\nTop: information about the selected protein. Bottom: new localization selection box rendered in the PPI viewer when clicking on the protein circle (http://cell.dallago.us/ppi?p=Q9NR71).\n\n(1) Top-left controls of PPI viewer; (2) top-left controls of map viewer; (3) top-center search panel of PPI/map viewer; (4) top-right layer control on PPI/map viewer.\n\nTo facilitate the retrieval of proteins and their interacting partners, CellMap provides basic search functionalities. Users can search for proteins based on their UniProt identifiers, by their gene identifiers or by their protein names. When performing the search, the page renders a grid containing boxes, each representing a different protein (Figure 7). Inside the boxes, the UniProt identifier for the protein that matched the search criterion is displayed. Starting on the top-right of every box a smaller colored square for each compartment is displayed in which that protein is localized. For proteins annotated to be in a single compartment, the border of the outer box (representing one protein as indicated by the UniProt ID in the center of the box) will get the color of that compartment (2nd box in Figure 7). Clicking on one of the colored squares will filter results based on the compartment represented by that color. In the bottom-right of each box, the total number of PPI partners are annotated.\n\nThe screenshot of this section of the home page shows four proteins that match the search criterion “foxo” either by their UniProt identifier, primary gene name or primary protein name. The protein boxes contain the UniProt identifiers of the matched proteins (center) and display the number of interaction partners (bottom-right) and several color-filled boxes graphically representing the localizations reported for the matched proteins (top-left).\n\n\nDiscussion\n\nSome CellMap functionality is exemplified by a heat shock protein (HSPA4; Heat shock 70 kDa protein 4, UniProt identifier P34932) with many interaction partners (338, according to HIPPIE, http://cbdm-01.zdv.uni-mainz.de/~mschaefer/hippie/query.php?s=HSPA4) in different compartments. The objective was to showcase how CellMap can simplify PPI hairballs. We visualize the same PPI network using CellMap (Figure 8A) and Cytoscape1 in the form of the Cytoscape.js version used by HIPPIE (Figure 8B) and the Cytoscape desktop version (Figure 8C).\n\nNone of the three viewers solves the PPI hairball problem completely. Without zooming in, the information density for 338 protein pairs is too high to be helpful. HIPPIE’s layout for Cytoscape.js (Figure 8B) clearly improves over the standard Cytoscape desktop version (Figure 8C) by centering the view around HSPA4, the protein of interest. In CellMap (Figure 8A) the biologically relevant differences between pairs from the same and from different compartments remain visible.\n\nPPI hub in CellMap (A), Cytoscape.js (B) and Cytoscape desktop (C). For HSPA4 (Heat shock 70 kDa protein 4, UniProt identifier P34932), we show some of the PPIs known (according to HIPPIE HSPA4 has 338 interaction partners). We chose this as one example of a protein with many more PPIs than the average protein (“PPI hub”). The figure compares how three different PPI viewers cope with the HSPA4 network: (A) CellMap (http://cell.dallago.us/protein/P34932), (B) HIPPIE’s Cytoscape.js visualizer and (C) the desktop version of Cytoscape. Proteins in CellMap are represented as colored dots on the map (image) of the cell, and upon selecting the protein of interest an overlay of edges is drawn. In Cytoscape and Cytoscape.js, proteins are represented as nodes containing a label (protein name as UniProt identifier), and edges are directly inferred from the data. The Cytoscape.js visualization was taken directly from HIPPIE. The Cytoscape network was automatically drawn upon loading the HIPPIE dataset and selecting the protein of interest and it’s direct neighbors.\n\nBy using a biologically relevant dimension (protein localization), instead of drawing nodes in positions based on edge weight (force layout of Cytoscape), some aspects of the protein and its partners become obvious at first glance, e.g. that HSPA4 interacts with many nuclear and cytoplasmic proteins, as well as with proteins that are secreted (extra-cellular) and located in the Endoplasmic Reticulum (ER, Figure 8). This may suggest the hypothesis HSPA4 to be an important hub involved in process spanning across compartments. Such a hypothesis is presented in our supplementary material (Figure SOM_1), where we analyze the visualization of the FOXO3 protein through CellMap.\n\nOne disadvantage of CellMap over the Cytoscape.js view is that the protein identifiers are not visible at all on the static image (protein identifiers become visible through mouse-over events in CellMap). However, in the image shown (Figure 8) the Cytoscape.js names also remain unreadable. Another problem with CellMap are the numbers displayed on edges (experimental reliability of the PPI as given by HIPPIE). In our view, this information is extremely important to look at interactions, but we are still lacking a more sophisticated mechanism to visualize these numbers.\n\nCellNetVis8 is a recent tool that also connects localization with PPI networks. It emphasizes the way PPI networks are laid out through the adaptation of a so-called force-directed layout (using the tool While). Although CellMap and CellNetVis are founded on a similar idea, user experience and focus differ importantly. For instance, CellMap can be driven by data from users that define the number of compartments on a map, and provide localizations. In contrast, CellNetVis uses a fixed subset of compartments and an ad hoc diagram for the cell. Additionally, CellMap comes with out of the box data for the human proteome and allows the community to grow the tool by enriching datasets (images and localizations), whereas CellNetVis has a per-use approach, allowing to visualize networks stored in specialized XGMML files. Another unique aspect of CellMap is the openness to introduce further biologically meaningful dimensions (beyond location such as time or pathways) that increase the usefulness of PPI visualization tools to create new testable hypotheses.\n\n\nConclusions\n\nCellMap is a prototype providing a portal exploring the idea of using protein subcellular location as the basis to construct more complete visualizations of biological data, such as protein-protein interactions (PPIs). Using this paradigm, we claim that additional information, such as pathways, can be layered on top of the current visualization of subcellular location to potentially generate meaningful biological insights. The source code for the portal is publicly available and an instance of the portal with location data from a previous publication about the subcellular localization of the human proteome5 and protein-protein interaction data from HIPPIE6 (http://cbdm-01.zdv.uni-mainz.de/~mschaefer/hippie) is running at http://cell.dallago.us. The visualization tool is written in JavaScript, thereby tapping into a very large user base for customized extensions and modifications. With the release of the prototype, we aim at creating a user base and awareness of the tool, ultimately collecting precious feedback from experimentalists and technical users alike.\n\n\nAbbreviations\n\n2D: two dimensions, API: Application Program Interface, ID: identifier, JSON: JavaScript Object Notation, PPI: protein-protein interaction, ROI: region of interest.\n\n\nSoftware availability\n\nThe CellMap prototype is released as open source software under the GNU General Public License v3.0. Documentation, source code and viewer are available at https://github.com/sacdallago/cellmap. Archived source code as at the time of publication is available at https://doi.org/10.5281/zenodo.9043249. An example of use with protein localization data from a recent publication5 and from the HIPPIE database of protein-protein interactions6 is available at http://cell.dallago.us.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the German Research Foundation (DFG) and the Technical University of Munich, within the funding programme Open Access Publishing.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThanks primarily to Tim Karl, but also to Guy Yachdav (all TUM) for invaluable help with hardware and software; to Inga Weise (TUM) for support with many other aspects of this work; to Dr. Luisa Jiménez-Soto (Max von Pettenkofer-Institut) for helpful comments on the manuscript; the LRZ Compute Cloud team for hosting the webserver; to Rolf Apweiler (UniProt, EBI, Hinxton), Amos Bairoch (CALIPHO, SIB, Geneva), Ioannis Xenarios (Swiss-Prot, SIB, Geneva), and their crews for maintaining excellent databases and to all experimentalists who enabled this analysis by making their data publicly available.\n\n\nReferences\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChaurasia G, Malhotra S, Russ J, et al.: UniHI 4: new tools for query, analysis and visualization of the human protein-protein interactome. Nucleic acids res. 2009; 37(Database issue): D657–D660. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMa DK, Stolte C, Krycer JR, et al.: SnapShot: Insulin/IGF1 Signaling. Cell. 2015; 161(4): 948–948.e1. PubMed Abstract \| Publisher Full Text\n\nOfran Y, Yachdav G, Mozes E, et al.: Create and assess protein networks through molecular characteristics of individual proteins. Bioinformatics. 2006; 22(14): e402–e407. PubMed Abstract \| Publisher Full Text\n\nRamilowski JA, Goldberg T, Harshbarger J, et al.: A draft network of ligand-receptor-mediated multicellular signalling in human. Nat Commun. 2015; 6: 7866. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlanis-Lobato G, Andrade-Navarro MA, Schaefer MH: HIPPIE v2.0: enhancing meaningfulness and reliability of protein-protein interaction networks. Nucleic Acids Res. 2017; 45(D1): D408–D414. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe UniProt Consortium: UniProt: the universal protein knowledgebase. Nucleic Acids Res. 2017; 45(D1): D158–D169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeberle H, Carazzolle MF, Telles GP, et al.: CellNetVis: a web tool for visualization of biological networks using force-directed layout constrained by cellular components. bioRxiv. 2017. Publisher Full Text\n\nDallago C: CellMap: open software for PPI and protein localization visualization in JavaScript. Zenodo. 2017. Data Source" }	[ { "id": "27544", "date": "13 Nov 2017", "name": "Sandra Orchard", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a tool for visualising PPI networks in the context of the subcellular localisation of the searched protein.\nI have twice tried to review this paper and both times the http://cell.dallago.us link gave me a MongoDB error. I have therefore had to review the paper without being able to view the tool. This is not satisfactory. I was unable to test the conclusions about the tool and its findings.\n\nThe tool uses a static interaction compilation database (HIPPIE) as the source of PPIs. Did the authors not consider using the PSICQUIC web service, which gives the users considerably more options as to where to source their PPI data from, and also allows the visualisation of protein-small molecule interactions and also potentially the site of action of protein-drug interactions, also available via PSICQUIC. It would also allow the data to be as up to date as the latest release of each database, which will be more frequent than releases of HIPPIE.\n\nI am not clear where the subcellular location data comes from. This may be obvious to regular users of CellMap but not to me, an should be stated in the paper for other user who do not know this.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "3208", "date": "27 Nov 2017", "name": "Christian Dallago", "role": "Author Response", "response": "Dear Dr. Orchard,Thank you for your valuable input on the tool!With regard to point 1: we apologize for the broken database connection, unfortunately, the deployment system missed that flag and thus didn’t restart the service. We have fixed the issue and the website is now running. Up until now, I have not identified any other issues that could prevent the web server to run properly.With regard to point 2: the software presented in this paper has a dual-purpose. On the one hand, we want to give the ability to discover protein localization and protein-protein interaction from two known sources (HIPPIE for PPI, and subcellular localization from a publication, which describes localization for the human proteome based on a consensus of experimental data and state-of-the-art prediction models (http://doi.org/10.1038/ncomms8866)). On the other hand, we want to propose a system that can be reused on user-defined data (as long as it complies with the format the visualization tool digest, as from Figure 1) and be integrated as JavaScript visualization tool in different portals. For now, we would like to avoid having a direct integration of the portal with external tools via, for example, API calls. In an upcoming version of the portal, we will offer scripts to populate the database from different sources for the two data entities (protein localization and interaction).PSICQUIC generates interaction data on-demand, which can later be downloaded. Obtaining the data requires some time: a user input one specific protein identifier, selects the databases to use to collect interaction data, submits a cluster job and finally gets access to the data. Searching for protein P45381 identified 80 interactions in all online databases. After several hours, the job was not finished, so we decided to lower the number of databases to fetch information from. Reducing the number of databases produced results quickly. The results page of PSICQUIC presents a table of interactions and visualizes a graph, which we could not load due to lack of compatibility with the Chrome browser. We believe it would be interesting to present CellMap at the level of this resource and will contact the authors of the tool to discuss what the best idea in this regard would be. Fetching the data from PSICQUIC as it is now and putting it into the portal requires to also normalize the PSICQUIC data and map it to protein localization data. Writing a parser for the PSI-MITAB tables is straightforward, the normalization and mapping of identifiers should occur externally to CellMap. We will create a guide on how this can be done in the next days and put it on the landing page of CellMap.Integrating protein-molecule data and displaying these entities meaningfully is an interesting idea for the future development of the CellMap tool.With regard to point 3: the data about protein localization stems from a publication of our group (http://doi.org/10.1038/ncomms8866). The data on protein subcellular localization for humans published through this paper was the starting point for the development of CellMap. In the current manuscript, we focused more on describing the visualization tool, rather than going into detail about how the localization data was retrieved (which in this case is by building a consensus over experimental (where available) and predicted localisations for 6 subcellular compartments). This is again because we didn’t want to develop a tool around this specific data source, but rather offer the possibility to change the origin for the localization data in the future.We appreciate the suggestions for further data sources and data entities that can be used and integrated into CellMap. In upcoming releases, we will make sure to offer a bigger variety of data sources and scripts to populate and update the information on protein subcellular localization, and protein-protein interaction data used by the visualization tool. Additionally, we will contact the authors of PSICQUIC to discuss if it would be possible to integrate CellMap in the results page of a cluster job.Best regards,Christian Dallago, Tatyana Goldberg & Burkhard Rost." } ] }, { "id": "29754", "date": "18 Jan 2018", "name": "Augustin Luna", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe tool provides an interesting feature to help declutter visualizations of biological networks using localization information. Some comments:\nIt would be good if the names of the used databases was stated in the last paragraph of the introduction.\n\nThe tool would be more intuitive for new users, if it provided descriptions the various colors used on the site with the same explanation as in the paper. For example, the colored boxes that represent localizations in the search results and the dot colors used for the protein visualization on the cell map.\n\nIt is unclear from the paper all the types of interactions might be shown in the represented networks.\n\nAlso, it is unclear from the paper, what happens to the network visualization in the cases where the identified proteins are present in multiple locations.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3380", "date": "29 Jan 2018", "name": "Christian Dallago", "role": "Author Response", "response": "Dear Dr. Luna,Thank you very much for your input on our work.We have submitted a new version of the manuscript, which should address points one and four of your comment.As to the second point: we have created a new feature item for our next release that displays a button on the map viewer to display a modal with the legend. As of now: a legend is available by scrolling down to the second half of the page in the map or ppi viewers (e.g. http://cell.dallago.us/map/573c87c182a9e1ae1e37d08e?p=P04637 ) and expanding the \"Legend\" tab. We understand that this can be overseen and improved, therefore we thank you for the input.As to the third point: in this manuscript, we focus on discussing the software implementation and visualization abilities of CellMap, rather than the data sources used in the example deployment hosted on http://cell.dallago.us. More information about the types of interactions reported by the HIPPIE data source can be found in the latest paper describing HIPPIE (http://nar.oxfordjournals.org/content/early/2016/10/28/nar.gkw985) and directly on the HIPPIE information page (http://cbdm-01.zdv.uni-mainz.de/~mschaefer/hippie/information.php#sources).Please, feel free to suggest any other changes to both our manuscript and tool.Best regards,Christian Dallago." } ] } ]	1	https://f1000research.com/articles/6-1824
https://f1000research.com/articles/6-784/v1	06 Jun 17	{ "type": "Method Article", "title": "A multi-tool recipe to identify regions of protein-DNA binding and their influence on associated gene expression", "authors": [ "Daniel E. Carlin", "Kassi Kosnicki", "Sara Garamszegi", "Trey Ideker", "Helga Thorvaldsdóttir", "Michael Reich", "Jill P. Mesirov", "Kassi Kosnicki", "Sara Garamszegi", "Trey Ideker", "Helga Thorvaldsdóttir", "Michael Reich", "Jill P. Mesirov" ], "abstract": "One commonly performed bioinformatics task is to infer functional regulation of transcription factors by observing differential expression under a knockout, and integrating DNA binding information of that transcription factor.\n\nHowever, until now, this this task has required dedicated bioinformatics support to perform the necessary data integration. GenomeSpace provides a protocol, or “recipe”, and a user interface with inter-operating software tools to identifying protein occupancies along the genome from a ChIP-seq experiment and associated differentially regulated genes from an RNA-Seq experiment. By integrating RNA-Seq and ChIP-seq analyses, a user is easily able to associate differing expression phenotypes with changing epigenetic landscapes.", "keywords": [ "ChIP-seq", "RNA-Seq", "transcription factor", "histone modification", "epigenetics", "regulation", "differential expression", "data integration" ], "content": "Introduction\n\nThe genetic make-up of an organism plays a key role in gene regulation, especially during early cell differentiation and development. We can observe this phenomenon in siblings who possess different eye and hair color as a result of differing genetic code. However, epigenetic mechanisms, such as histone modifications, transcription factor binding and DNA methylation, also contribute to the complexity of individuals’ phenotypes as is observed in identical twins who possess the same genetic code while having slightly different features. Phenotypic differences associated with disease and varying stages of development have been mapped to changing patterns in gene regulation; and phenotype can often be attributed to a changing epigenetic landscape rather than hard-coded genetic features.\n\nIn order to decode these epigenetic differences, biologists often turn to an analysis based on two experimental assays; RNA sequencing (RNA-Seq) (Nagalakshmi et al., 2008; Wilhelm et al., 2008), which quantifies the amount of (usually messenger) RNA in a cell, and Chromatin Immuno-precipitation sequencing (ChIP-seq) (Johnson et al., 2007; Robertson et al., 2007), which shows where a particular protein binds the genome. Commonly, this protein is expected to have some influence on the mRNA expression of nearby genes (i.e., it is a transcription factor). Thus, by knocking out the gene that codes for the DNA binding protein and observing changes in mRNA expression, the biologist can infer the direct effect of the protein on expression.\n\nWhen analyzing genomic data, today’s computational biologist may utilize a variety of different tools specific to each step of their analysis process. Not only must they be able to create the perfect marriage between the type of data and the tool, but they must be able to correctly manipulate the output, both for interpretation and for format conversion between tools. For the non-programming biologist, smooth integration of many of these tools is provided through GenomeSpace (Qu et al., 2016, www.genomespace.org) and its user-friendly “recipes” (recipes.genomespace.org). GenomeSpace is a web-based visual workbench that supports a diverse range of bioinformatics tools and data resources popularly used in genomic analyses. Because GenomeSpace provides the ability to reformat data as it moves between software tools, one can create easy to use step-by-step workflows specific to a given analysis task. We refer to these published workflows as “recipes”.\n\nWe present one such recipe, currently available in GenomeSpace, which identifies differentially expressed genes between two samples, and compares that gene list with differential transcription factor occupancy from a ChIP-Seq experiment. This recipe is designed to elucidate which DNA-protein binding events are responsible for an observed change in mRNA expression. By identifying protein occupancies throughout the genome and comparing them to observed differences in mRNA expression, we can support hypotheses of functional regulation.\n\n\nMethods\n\nThis recipe takes as input the aligned reads from a differential RNA-seq transcription factor knockout experiment, and aligned reads from a ChIP-Seq experiment for the transcription factor that was knocked out. The output is a visualization of the genomic regions containing both differentially expressed genes and a binding site for the transcription factor. Since all tools used in this recipe are hosted remotely, running the recipe has no system requirements beyond an internet connection. We describe the individual steps of the recipe here.\n\nWe start by obtaining a reference genome matching our model organism and aligning RNA-seq reads from two or more conditions (e.g. experimental and control) and ChIP-Seq reads from at least two samples, an input control and an experiment. In ChIP-Seq, the input control is a sample that has been run through all of the same preparatory and sequencing steps as the experiment, except for the antibody binding. This controls for the natural background of reads that are not selected by the binding of the target protein. Both RNA-seq and ChIP-Seq read data are uploaded to GenomeSpace in the BAM (Binary sequence Alignment MaP) format and the reference genome in the GTF (Gene Transfer Format).\n\nWe next perform differential expression analysis using GenePattern (Reich et al., 2006, genepattern.broadinstitute.org), which can be launched from the GenomeSpace user interface. We use GenePattern’s Cuffdiff module to identify genes with differential expression between samples, measured by their FPKM (Fragments Per Kilobase of transcript per Million mapped reads) value. For each condition, we input the read data for an individual sample followed by the GTF reference genome. The output of the differential analysis is exported to GenomeSpace in Cuffdiff’s tabular format.\n\nWe next launch Galaxy (Afgan et al., 2016; Giardine et al., 2005, galaxyproject.org), again available through the GenomeSpace interface, and import RNA-seq reads from both conditions along with a file containing differential expression for each gene. This data is directly available through GenomeSpace. Using a Galaxy workflow, we filter genes that are significantly (q-value < 0.05) differentially expressed between the experiment (in this case a knockout) and control samples and extract their chromosome number, gene region start, gene region end, and gene symbol. Next we use Galaxy’s SAMtools (Li et al., 2009) Filter subtool, which extracts this data from the original RNA-seq reads in the BAM format. We convert the BAM files to the bigWig format so that they can be viewed in the Integrative Genomics Viewer (IGV) (Robinson et al., 2011; Thorvaldsdottir et al., 2013).\n\nNext, we use GenomeSpace to import the ChIP-seq files from both the input control and experimental samples to Galaxy. Using Galaxy’s MACS2 (Feng et al., 2012) callpeak subtool, we obtain a bedGraph file containing peak-enrichment data of both our experimental and input control files. Additionally, we use the MACS2 callpeak tool to identify differential peaks along the genome, indicative of transcription factor binding sites, and output this data as a bedGraph file. The two bedGraph files are converted in Galaxy to the bigWig format for visualization in IGV.\n\nWe next launch IGV through the GenomeSpace user interface. We select the appropriate reference genome included in IGV, and load all gene expression and peak-enrichment Bigwig files from GenomeSpace. Tracks are then scaled by group so their track heights are adjusted accordingly for better visualization.\n\n\nUse case\n\nWe applied the recipe described above to an example dataset from Laurent et al. (2015), accession GSE6328, from NCBI’s Gene Expression Omnibus (GEO) database (Barrett et al., 2013; Edgar et al., 2002). We can identify the interplay between the epigenetics and transcriptomics of mouse embryonic stems cells by observing how the binding of the transcription factor, Prep1, influences gene expression. Prep1 is known for its contribution in embryonic development (Laurent et al., 2015). In comparing genome-wide maps of mouse embryonic cells expressing Prep1 to those that do not, we can identify potential target genes that are being differentially regulated by these binding events. One such example of this is illustrated in Figure 1. Here, the transcription factor binding site has been identified and shown to up-regulate the expression of the gene Igf2.\n\nThe left panel illustrates the binding of the Prep1 transcription factor. In the right panel, we see the up-regulation of the gene, Igf2, as a result of this binding event.\n\n\nVariations of this recipe\n\nThis recipe can be used, not only to identify the regulation of genes by transcription factor binding, but also to identify any epigenetic mechanism that can be analyzed by ChIP-sequencing. For example, we can identify regions in the genome where histone modifications have occurred, and match those regions to observed changes in expression presumably resulting from the histone modifications. However, we must consider the nature of the data when selecting parameters in the MACS2 tool in Galaxy. For example, when performing peak enrichment on histone modification occupancies, a user must select an advanced option to include broader regions, since histone modifications are represented by a much broader peak area along the genome.\n\n\nData availability\n\nThe original ChIP-seq and RNA-seq data of this experiment have been deposited in GEO, with accession number GSE63282. The recipe providing all the detailed steps and corresponding videos associated with this process is accessible at: http://recipes.genomespace.org/view/69.", "appendix": "Author contributions\n\n\n\nDC, KK and SG designed the software protocol. KK and SG prepared a first draft of the manuscript. DC and JM finished the manuscript. JM, TI, and HT oversaw the administration and management of this project.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by the National Human Genome Research Institute, NIH U41HG007517.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nAdditional members of the Mesirov Lab, specifically Ted Liefeld and Clarence Mah at the University of California- San Diego, aided in the testing and editing of this protocol.\n\n\nReferences\n\nAfgan E, Baker D, van den Beek M, et al.: The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. Nucleic Acids Res. 2016; 44(W1): W3–W10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarrett T, Wilhite SE, Ledoux P, et al.: NCBI GEO: archive for functional genomics data sets--update. Nucleic Acids Res. 2013; 41(Database issue): D991–D995. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res. 2002; 30(1): 207–210. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFeng J, Liu T, Qin B, et al.: Identifying ChIP-seq enrichment using MACS. Nat Protoc. 2012; 7(9): 1728–1740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGiardine B, Riemer C, Hardison RC, et al.: Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 2005; 15(10): 1451–1455. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJohnson DS, Mortazavi A, Myers RM, et al.: Genome-wide mapping of in vivo protein-DNA interactions. Science. 2007; 316(5830): 1497–1502. PubMed Abstract \| Publisher Full Text\n\nLaurent A, Calabrese M, Warnatz HJ, et al.: ChIP-seq and RNA-seq analyses identify components of the Wnt and Fgf signaling pathways as Prep1 target genes in mouse embryonic stem cells. PLoS One. 2015; 10(4): e0122518. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNagalakshmi U, Wang Z, Waern K, et al.: The transcriptional landscape of the yeast genome defined by RNA sequencing. Science. 2008; 320(5881): 1344–1349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQu K, Garamszegi S, Wu F, et al.: Integrative genomic analysis by interoperation of bioinformatics tools in GenomeSpace. Nat Methods. 2016; 13(3): 245–247. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReich M, Liefeld T, Gould J, et al.: GenePattern 2.0. Nat Genet. 2006; 38(5): 500–501. PubMed Abstract \| Publisher Full Text\n\nRobertson G, Hirst M, Bainbridge M, et al.: Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing. Nat Methods. 2007; 4(8): 651–657. PubMed Abstract \| Publisher Full Text\n\nRobinson JT, Thorvaldsdóttir H, Winckler W, et al.: Integrative genomics viewer. Nat Biotechnol. 2011; 29(1): 24–26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThorvaldsdóttir H, Robinson JT, Mesirov JP: Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 2013; 14(2): 178–192. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilhelm BT, Marguerat S, Watt S, et al.: Dynamic repertoire of a eukaryotic transcriptome surveyed at single-nucleotide resolution. Nature. 2008; 453(7199): 1239–1243. PubMed Abstract \| Publisher Full Text" }	[ { "id": "23943", "date": "11 Jul 2017", "name": "Isha Sethi", "expertise": [ "Reviewer Expertise Next-Generation Sequencing", "Genomics", "Epigenomics", "Transcriptomics", "Chromatin" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have made a pipeline integrating differential RNA-Seq expression analysis with ChIP-Seq analysis and implemented it through the GenomeSpace platform. Though as mentioned by the authors in the paper: this is a commonly performed bioinformatic task, their aim is to make this integrated analysis easily accessible to non-bioinformatic users. For this purpose their workflow on the web-based workbench involves integrating multiple tools like Cuffdiff module in GenePattern (for Differential RNA-Seq analysis) with MACS2 in Galaxy (for ChIP-Seq analysis).\n\nThough the workflow presented by the authors seems easy to use by any biologist, it also appears to be severely limited not just in its scope of application but also in its choice of tools which are hardcoded. For example the authors use \"CuffDiff\" for Differential RNA-Seq expression analysis. The authors do not state why they chose this particular method or even why they chose its GenePattern module and not the Galaxy implementation. Though admittedly this is a popular tool and has the advantage of transcript level analysis, it also suffers from known limitation of underestimating the number of differential genes. Other count based method like DESeq2 tool (also implemented in Galaxy) might be better suited for most gene-level differential RNA-Seq analysis. Also, the authors do not clearly explain why their workflow is better or easier for a biologist to implement than using the same tools through Galaxy directly (which has been made for a non-coding biologist). I would argue that working directly on Galaxy even if slightly more complicated would be more rewarding to users as it offers not just greater flexibility of tools but also the option to select different parameters than default.\n\nHence in conclusion, to make this manuscript better the authors should 1) provide a clearer explanation for their choice of tools and why is it easier/better to use their pipeline than the same tools on Galaxy directly, 2) If possible the authors should try to expand their workflow to provide a greater flexibility to the user to choose their tools for RNA-Seq and ChIP-Seq analysis.\n\nIs the rationale for developing the new method (or application) clearly explained? Partly\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "3065", "date": "28 Sep 2017", "name": "Jill Mesirov", "role": "Author Response", "response": "We greatly appreciate the reviewers' comments pointing out that the \"Variations of the Recipe\" section of this manuscript did not discuss alternative tools. The GenomeSpace recipes are subjective in that they use tools with which the authors are most familiar and comfortable. However, we do recognize that there are, of course, other options. We will revise the manuscript (and the recipe) to include some of the other options that could be used. In the past that has been our custom and we apologize that we did not catch the lack of this information in this recipe. In fact, on the best practices page of our website - http://recipes.genomespace.org/page/best-practices-for-creating-recipes we suggest authors supply alternatives. We would invite both the reviewers and other readers of the manuscript to contribute versions of this recipe using their preferred tools and approaches. GenomeSpace is a community project and contribution of recipes is both welcome and encouraged." } ] }, { "id": "24958", "date": "22 Aug 2017", "name": "Andrew D Sharrocks", "expertise": [ "Reviewer Expertise Gene regulation", "including RNAseq and ChIPseq analysis" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a workflow (which they refer to as a “recipe”) for the integration of RNA-Seq and ChIP-Seq experiments to find associations between genomic binding of TFs and their potential direct effects on the mRNA expression using the web-based work-bench GenomeSpace. The workflow enables integration of differential gene expression analysis following transcription factor knockdown (RNA-seq data) with binding data for the same transcription factor (ChIP-seq data). While there are many useful pipelines available for analysing and integrating sequencing data, GenomeSpace and its associated “recipes” make the analysis and integration of the data less daunting for a biologist with little or no programming experience.\n\nOverall this “recipe” will likely be useful for biologists. However, we would like to make a few comments:\n\n1. The authors do not make it clear whether the user has to pre-align the files to the reference genome before uploading them to GenomeSpace or the alignment itself can be done via their recipe. If not then it will be useful to provide the necessary tools and guidance required for alignment given that aligning the reads to genome is one of the most memory and time consuming steps.\n\n2.The authors use the Cuffdiff module from the Cufflinks package to perform differential expression. Although the goal of this recipe is providing user-friendly and simplified workflow for integration of data the authors should mention the advantages of alternate tools such as EdgeR and DeSeq for identifying differential expression. These tools are available in GenePattern therefore it will be sensible to provide the user with all options. Especially since these tools are known to have better normalisation techniques and perform a more robust and reliable identification of differentially expressed genes compared to Cuffdiff.\n\n3. The authors should not confuse the Enriched peak data obtained by using the MACS callpeak tool on the ChIP data and its input with Differential peaks. Differential peaks are obtained between two experimental conditions and not between the ChIP experiment and its input. This language can be misleading especially for beginners.\n\n4. A flowchart of the analysis steps in the paper would be highly useful to get started.\n\n5. Figure 1 is not entirely clear as presented. It is not clear why two separate panels are provided rather than a single panel that shows the location of the ChIP-seq peaks relative to the gene expression changes. Also, indicating what the colours represent in the gene expression data. The track labelling on the left is also not clear, and presumably “overlay” is the RNAseq data and “peaks” the ChIP-seq data. While the current labelling is presumably driven by the naming of the original files, better labelling is suggested in the context of this figure so it is clear to the reader. Finally, looking at the coordinates provided, we are not convinced that this is a good example to provide. The TF binding event Appears to be over 2.5Mb from the TSS of the putative target gene, meaning that any links here would be fairly low confidence. This would of course become more obvious if displayed on a single panel.\n\n6. To make the “recipe” really useful, it would be good to have outputs beyond simple genome browser views. Having a tabular output of differentially expressed genes and the relative location(s) (and coordinates) of any binding peaks for the transcription factor in question would be useful to have.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Partly\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-784
https://f1000research.com/articles/6-1663/v1	08 Sep 17	{ "type": "Review", "title": "Blinding in trials of interventional procedures is possible and worthwhile", "authors": [ "Karolina Wartolowska", "David Beard", "Andrew Carr", "David Beard", "Andrew Carr" ], "abstract": "In this paper, we have used evidence from our earlier review of surgical randomised controlled trials with a placebo arm to show that blinding in trials of interventional procedures is feasible, and that many creative methods can be used to make the active and the placebo procedure as similar as possible. We give examples of ingenious strategies used to simulate the active procedure and make the placebo control indistinguishable from the active treatment. We discuss why it is important to blind of patients, assessors, and caregivers and the types of bias that may occur in interventional trials. Finally, we describe the benefits of blinding, from the obvious ones such as avoiding bias, as well as less evident benefits such as avoiding patient drop out in the control arm.", "keywords": [ "blinding", "RCT", "randomised controlled trials", "placebo", "surgery", "procedures", "non-pharmacological" ], "content": "Introduction\n\nThe aim of a trial is to produce unbiased evidence. As randomised controlled trials (RCTs) with a placebo arm control for many types of bias, they are regarded as the most reliable method of demonstrating treatment efficacy and provide the highest level of evidence1. RCTs of interventional procedures are rare2–5, partly because they are challenging6; however, they are not impossible to perform, even if they involve a placebo arm7. In this paper we will discuss why trials should be blinded and summarise the methods which have been used in the published placebo-controlled trials of interventional procedures to achieve blinding.\n\nBlinding in interventional trials is often necessary because nowadays many procedures are performed to reduce pain and improve function and quality of life. Pain, function and quality of life are sometimes regarded as preferable outcome measures because they reflect patients’ needs and point of view8. However, as these outcomes depend on patients’ subjective perception, they are prone to bias and may lead to an exaggerated treatment effect in open-label trials9,10. Using subjective outcomes in an open-label study undermines its internal validity because it is not possible to determine how much of the reported effect is related to the investigated treatment and how much is related to bias.\n\n\nBlinding of patients, surgeons, outcome assessors and caregivers\n\nBlinding means concealing the treatment allocation from patients and any other people involved in the trial who may bias the results of the trial by knowing which groups the patients were randomised to.\n\nBlinding of patients prevents reporting bias in patient-reported measures. For example, it has been demonstrated that non-blinded patients exaggerate the effects size by 0.56 standard deviations and that the effect is even larger in trials on interventional procedures, such as acupuncture11,12. This bias may be caused by patients’ expectations of treatment effect and information given to them before the treatment13. Patients may also report symptoms depending on their “hunches” about treatment being effective or they may give answers they believe are “correct” or expected from them, for example, because it would have been impolite not to report improvement11. Therefore, it has been suggested that patients should be blinded whenever possible11.\n\nBlinding of patients also reduces adherence bias, i.e. patients in the control group not following the protocol/treatment. It may also prevent so called “contamination of the control group”, i.e. seeking additional treatment/help outside the trial and receiving concomitant treatment. Blinding improves patient retention in the trial. Risk of attrition in blinded trials is about 4% whereas in non-blinded trials it is 7%11. Specifically in placebo-controlled surgical trials, subject retention is often reported as “excellent”14, and in our analysis the withdrawal rate was low (4%) and comparable between the treatment and the placebo arm15.\n\nUnlike drug trials, in which the physician gives a tablet prepared somewhere else, the surgeon has to perform a specific procedure considered to be therapeutic; therefore, blinding of surgeons may not always be possible.\n\nThere have been attempts to blind surgeons, for example a surgeon inserted a catheter under fluoroscopic guidance and handed over the procedure to a technician who delivered the radiofrequency energy (or not) according to the allocation16. In other trials, a palatal implant delivery system was prepared by the manufacturer to either contain the implant or not, which allowed for blinding of surgeons17,18.\n\nIn 81% of placebo-controlled surgical trials both patients and assessors were blinded15. It has been demonstrated that, non-blinded assessors of subjective outcomes cause less bias in trials than non-blinded patients reporting their symptoms19. Blinding of assessors prevents observer-related bias, detection bias, and the Pygmalion effect. The Pygmalion effect refers to a situation when investigators looking for a particular response are predisposed to interpret the result in a way that shows the response they expect even if it is objectively absent. For example, a study by Hrobjartsson and colleagues demonstrated that non-blinded assessors were over-optimistic and “over-rated” patients in the treatment group rather than “under-rated” patients in the control group20. In some trials the assessment was done by people not involved in the surgery, for example blinded researchers, staff at another hospital that they were operated on at21, or by pathologist blinded to the treatment allocation22.\n\nApart from blinding patients and assessors, it is important that caregivers and clinical/research staff do not know patient treatment allocation, because their behaviour and attitudes may influence patient responses23–25. Patient-clinician interaction plays an important role in treatment response, and patients in trials do better as they get more attention and time from clinical staff than patients receiving standard care26,27. Therefore, the interactions between patients and the trial team should be standardised so that the “treatment context” (similar attention from doctors, expectations, and settings) are comparable between the groups.\n\n\nStrategies used to maintain blinding in interventional placebo-controlled trials\n\nA placebo control is necessary if we want to know whether improvement is really caused by the investigated procedure. It compares the intervention of interest with a procedure that seems identical but does not involve the crucial element of believed to be “the cure”. The aim of a placebo arm is to control for effects of receiving treatment not specifically related to the investigated intervention.\n\nIt is often difficult to determine what is a specific and what is a non-specific effect in a trial27,28, and to disentangle placebo response from response bias or the effect of patient-doctor interactions29. It is beyond the scope of this review to discuss definitions of placebo1,29. Whether something is or is not a placebo depends on the intervention and chosen outcome variables1, but in order for blinding to be successful, the control procedure has to be as similar as possible to the investigated procedure27. Interventional trials differ from drug trials as they require access to the anatomical structure of interest; therefore, they involve a skin incision or an insertion of a scope.\n\nMany published surgical trials used general anaesthesia or heavy sedation, which made blinding easier because patients were unaware of the details of surgical procedures. In such trials, only the surgical wound had to be similar in both groups. Some studies did not add any placebo procedure but simply omitted part of interventional procedure, for example, in the trial by Stone and colleagues, all patients underwent a percutaneous coronary intervention and maximal medical therapy but only patients in the active arm also had percutaneous transmyocardial revascularisation30.\n\nWhen light sedation or local anaesthesia are used, surgical staff have to simulate the actual intervention to preserve the blinding. The complexity of a surgical procedure can make blinding challenging, and ingenious ideas are required to make the real and placebo interventions indistinguishable.\n\nIf a procedure requires open surgery, then it will leave an obvious mark where the incision has been made, which will have to be imitated in the placebo group. There have been very few trials involving full skin incision, in both the surgical and placebo arms. In the seminal trials on internal mammary artery ligation31,32 a skin incision was made to expose the arteries in all patients but no ligation was made in the placebo group. Similarly, Guyuron and colleagues used a skin incision to expose superficial nerves and muscles, which were cut during the active surgery but, in the placebo group, the integrity of these structures was maintained33. Trials investigating transplantation of dopaminergic neurones as a treatment for Parkinson’s disease not only required skin incision but also burr holes in the skull34,35.\n\nHowever, most of the published placebo-controlled surgical trials used minimally-invasive methods to access the structure of interest. For example, the placebo procedure involved laparoscopy but without ablation36, endoscopy without radiofrequency energy delivery37, bronchoscopy without radiofrequency energy delivery38, or bronchoscopy without valve placement39. Therefore, most of the studies required a small incision to mimic the portals created during the laparoscopy or arthroscopy, or to mimic the incision through which an intravascular catheter was inserted40. Interestingly, Sutton and colleagues used three incisions in both groups so that patients could not tell apart a diagnostic laparoscopy from a laparoscopic surgery; even though the third instrument port was not necessary in the placebo group41. Trials using endoscopy and bronchoscopy were even easier to blind as natural orifices were used to insert the scope, and the incision or actual procedure site was not visible to patients, caregivers, and assessors.\n\nTypically, the preparation for the placebo procedure and the active procedure was as similar as possible and immitated the visual, auditory, and physical cues42–45. In order to mimick the sounds, surgeons were required to talk through the procedure steps46, ask for instruments47,48, use suction48 or ask for a laser or other device to be activated, even though it was not used in the placebo group49–53.\n\nClinical staff performing the intervention were screened from the patients’ view54 either by a surgical drape52 or by arranging the operating room in a way that the patient could not see the procedure44. In the trial by Stone and colleagues, patients were heavily sedated and wore opaque goggles30. In a trial by Maurer and colleagues, the manufacturer delivered tools that looked identical but did not contain an implant, which allowed for blinding of patients and clinical staff18.\n\nSurgeons also attempted to immitate sensory cues, for example by manipulating the knee as if the actual arthroscopy were performed48, injecting saline to imitate tidal irrigation14, or by splashing saline on the knee to simulate lavage45. In a trial on meniscectomy, surgeons used a mechanised shaver (without the blade) pushing it firmly against the patella to simulate the sensations the patient would experience during the surgery48. In a trial on intragastric balloon for obesity, operators manipulated the endoscope as during the balloon insertion to create the sensation of resistance in the stomach53.\n\nEven smell during the surgery was imitated to make the placebo procedure indistinguishable from surgery. For example, in the trial by Deviere and colleagues there were concerns that patients could have known the allocation because the copolymer used in the active arm had a distinct smell55. In trials on vertebroplasty, a container with cement was opened during placebo procedure to help with blinding42,56.\n\nIt is important that the procedure used for blinding does not have any therapeutic effect. For example, the results of the vertebroplasty trials42,56 were criticised because the elements of placebo procedure could have had an effect on the reported pain, namely, a potential pharmacological anaesthesia due to injection of an anaesthetic into the facet capsule and periosteum57.\n\nOn the other hand, the procedure used for blinding may have diagnostic use, as with diagnostic laparoscopy36,41,58 or diagnostic laparoscopy with biopsy59. In the trial by Sihvonen and colleagues, all participants underwent diagnostic arthroscopy, but only after they had been confirmed to be eligible for inclusion in the trial was the envelope with the assignment opened and the assignment revealed to the surgeon48.\n\nMany trials specifically stated that the duration of procedure in the surgical and control arms were matched, either by immitating the elements of the surgical procedure or by keeping all patients in the operation room for the same duration of time34,38,45,47,48,60,61. However, in some trials, the placebo procedure was shortened in comparison to the actual surgery because it was believed it would have been ethically unacceptable to prolong the placebo intervention49,55.\n\nInterventional treatment often requires additional procedures, such as diagnostic scans or medication to prevent infection43,56,62, blood clots40, transplant rejection63, or epileptic fits35. For example, in the trial by Freed and colleagues, both groups received identical preoperative evaluation, intraoperative sedation and pain control, underwent two PET scans and a MRI scan, and received phenytoin35. In some trials, the same medication was given in both groups, whereas in others unnecessary treatment was omitted or imitated, for example by injecting saline instead of antibiotics64.\n\nThe active and placebo procedure have to be indistinguishable but they also have to be stable and standardised. Standardisation of the procedure itself may be difficult but is important because surgeons vary in their experience, and gain experience throughout the trial.\n\nSome of the changes observed in a trial may not be related to the treatment or the placebo intervention, but may be caused by the natural course of the disease, spontaneous remissions or fluctuations in the severity of symptoms or regression to the mean27,65. Some changes may be a result of just being in a trial either because of lifestyle changes that are part of the protocol such as self-monitoring using diaries or avoiding alcohol or due to so called “Hawthorne effect”, which refers to change in the behaviour when people, both patients and doctors, know that they are being observed27. Finally, it has been demonstrated that adhering to a protocol improves the performance of doctors, and that patients who adhere to treatment regimes have better outcomes66. Therefore, it is important to standardise pre- and post-operative care, and the explanations given to the patients. For example, in a trial by Sihvonen and colleagues all procedures were standardised and recorded on video; the post-operative care was also standardised, and all patients received the same exercise programme and walking aids48.\n\nMost trials blinded the assessors while the surgeon and other staff in the operating room were aware of the group assignment, and did not participate in further treatment, post-operative care or follow-up of the patient36,48,67. In a trial by Thomsen and colleagues, the post-operative care and assessment was done at a different hospital than the surgery21. In a trial by Cotton and colleagues a post-operative care was provided by the referring physician, who was blinded when deciding on treatment, and when this was not sufficient, by the evaluating physician at the study site (who was also blinded)67.\n\nThere are other types of bias that are specific to surgical trials. For example, in trials on upper gastrointestinal tract bleeding, the endoscopic procedure was not performed if the rate of blood loss was too fast, or the endoscopy was judged to be life-threatening and posed an unacceptable risk68–70. In other trials, some patients were excluded because they could not tolerate endoscopy, or due to anatomical conditions that made the surgery impossible to carry out (laser could not be aimed at the bleeding arteries)70. Alternatively, some patients were not included in a trial because they were no longer eligible, for example because the bleeding had stopped70 or they no longer reported the symptoms on the day of the study71.\n\n\nConclusions\n\nBlinding in trials of interventional procedures is possible and many creative methods have been used to maintain the blinding. Interventional procedures are challenging to blind, but the effort is worthwhile because of the obvious benefits, such as avoiding bias, as well as the less evident benefits, such as avoiding patient drop-out in the control arm.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work has received funding from the NIHR Oxford Musculoskeletal Biomedical Research Unit.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nGøtzsche PC: Is there logic in the placebo? Lancet. 1994; 344(8927): 925–6. PubMed Abstract \| Publisher Full Text\n\nMcLeod RS: Issues in surgical randomized controlled trials. World J Surg. 1999; 23(12): 1210–4. PubMed Abstract \| Publisher Full Text\n\nWente MN, Seiler CM, Uhl W, et al.: Perspectives of evidence-based surgery. Dig Surg. 2003; 20(4): 263–9. PubMed Abstract \| Publisher Full Text\n\nErgina PL, Cook JA, Blazeby JM, et al.: Challenges in evaluating surgical innovation. Lancet. 2009; 374(9695): 1097–104. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCook JA: The challenges faced in the design, conduct and analysis of surgical randomised controlled trials. Trials. 2009; 10: 9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFairbank J: Randomised controlled trials in surgery. Lancet. 1999; 354(9174): 257. PubMed Abstract \| Publisher Full Text\n\nWartolowska K, Judge A, Hopewell S, et al.: Use of placebo controls in the evaluation of surgery: systematic review. BMJ. 2014; 348: g3253. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "25898", "date": "20 Sep 2017", "name": "Ian A Harris", "expertise": [ "Reviewer Expertise Orthopaedics", "Epidemiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is a narrative overview of methods of blinding in trials of procedures, using the previous review by these authors as the source of data. It explains the rationale for blinding and gives examples (subgrouped) of how blinding was achieved in previous trials. The authors wisely avoid a detailed discussion of the placebo effect and concentrate on the need for blinding based on empirical evidence of bias resulting from not blinding. The paper reinforces the need for blinding in procedural trials which is a reasonable demand that is increasingly being heeded. In other words, the message is important. The paper is very well written. I only have minor comments, below:\nPage 2, second last paragraph, “crucial element of believed to be the cure” should read “crucial element believed to be the cure” The evidence of bias resulting from not blinding may be one sided. Opposing evidence should be discussed, such as Berkmann ND, The Empirical Evidence of Bias in Trials Measuring Treatment Differences1 which shows that the effect of blinding may even be in the opposite direction and the classic Schultz article from 19951 which gives a lower estimate of effect exaggeration from unblinding. There are several other summaries and meta-epidemiological studies in this area.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3340", "date": "30 Jan 2018", "name": "Karolina Wartolowska", "role": "Author Response", "response": "We are grateful for the comment and we have added an explanation that trials have limited external validity, as by controlling for confounding factors and standardising the trial conditions, they become very different from usual clinical practice. They are also liable to have study populations that are so unique that the results of the trial cannot be easily generalised. In the case of blinding, the magnitude of effect may be lower in a blinded trial than in everyday practice, because blinding causes uncertainty about treatment allocation. In clinical practice, there is usually no doubt as the patient-doctor relationship is based on trust and on the assumption that doctors offer patients the best available treatment. In an RCT, there is an inherent uncertainty as to which treatment group a patient has been allocated.(Enck et al., 2011) REFERENCESEnck, P. et al. (2011) ‘The placebo response in clinical trials: more questions than answers.’, Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 366, pp. 1889–1895." } ] }, { "id": "28006", "date": "04 Dec 2017", "name": "Harald Walach", "expertise": [ "Reviewer Expertise Research methodology", "clinical and other trials" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis review is, as its self-set goal goes, very well substantiated, balanced and timely. There is no factual correction in itself that is necessary in my view.\n\nHowever, I wish to give a few critical feedbacks to the authors that they may wish to integrate. I would prefer if they did, but in my view it is unfair to formally demand such an extension. However, the debate itself would profit.\n\nMy point is regarding the presuppositions of the current model the authors implicitly subscribe to, for instance when they say randomised trials “provide the highest level of evidence” (1st para). This is true, but only from a - debated – point of departure that accepts the hierarchisation of evidence as a given and true proposal. As it happens, we, and others, have debated this [1,2]. The hierarchisation of evidence accepts – without further reflection – that internal and external validity are compatible, linear concepts, where first internal evidence can be gleaned and is prior and superior to external validity. This I find an unsubstantiated point of view that needs at least acknowledgement. All the arguments in this paper address the improvement of internal validity, and as such they are very good.\n\nThe argument that “specific” effects have to be tested against “non-specific” or placebo-effects and therefore blinding is necessary also makes presuppositions that are not clarified and critically debated. The presupposition is that there is such a thing as separability of these effects. This separability argument is highly speculative, very little researched and where and when researched found to be wrong [3, 4]. One can, of course, try and disentangle various components of interventions. And especially where interventions are costly or fraught with side-effects this is even an ethical requirement, because else patients would be submitted to problematic procedures with little benefit or where other interventions might be just as good, all taken together. But one should be clear about the fact that this is not something that automatically gets rid of all problems in evaluating therapeutic procedures.\n\nTherefore, I think the arguments the authors present are valid within the context of improving internal validity of intervention trials that offer costly and potentially dangerous or problematic interventions. But I think the authors should be clear themselves that this is not a universally valid strategy and it would be wise to integrate some caveats and restrictions in their argument.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3339", "date": "30 Jan 2018", "name": "Karolina Wartolowska", "role": "Author Response", "response": "We are grateful for the comment regarding the hierarchy of evidence. We have rephrased the sentence so that it now reads “high level of evidence because they attempt to minimise bias due to confounding factors”. We have also added the suggested reference (Walach and Loef, 2015). We agree that RCTs have high internal validity because they are performed in a standardised way, and because they attempt to control for confounding factors. However, owing to the controlled conditions under which they are performed, RCTs often have low external validity. In this paper, we wanted to draw attention to the fact that surgical trials often use subjective outcomes, and are therefore prone to bias, which has to be minimised or controlled for. We agree with the Reviewer, that the “specific” and “non-specific” effects are difficult to separate or to unequivocally define. As surgical procedures are complex and consist of many elements, it is sometimes difficult to identify the crucial surgical element that is believed to be therapeutic. We have described the CSAW trial as an example. The aim of this trial was to investigate whether the improvement was due to change of anatomy (arthroscopic removal of a bony spur on the acromion) or it was not related to the change in anatomy and the spur removal was not necessary because a placebo procedure (arthroscopy only) results in similar change in pain and function. We wanted to draw attention to the fact that in the case of surgical trials, with their high costs and risks, one would expect to see a much larger effect after a surgical procedure than after a placebo or no treatment. We are familiar with the debate in the literature regarding specific and non-specific effects. In the context of surgical trials, by “specific effects” we meant changes directly related to change in anatomy, for example, a bony spur removal in the trial of arthroscopic shoulder decompression surgery for subacromial pain. All other possible factors resulting in improvement, such as the effect of arthroscopy, the placebo effect, influence of patient-doctor interactions, symptoms fluctuation as well as spontaneous improvement were regarded as “non-specific”. We also agree with Enck and colleagues that the effect in the placebo and active arms may not be additive; therefore, the improvement related to the crucial surgical element may not equal the difference between the effect in the active and placebo arms.(Enck et al., 2011) We would like to clarify that blinding and placebo control help to minimise bias and aid in the interpretation of trial results; especially if the outcomes are subjective and the symptoms fluctuate or may spontaneously improve.REFERENCESEnck, P. et al. (2011) ‘The placebo response in clinical trials: more questions than answers.’, Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 366, pp. 1889–1895.Walach, H. and Loef, M. (2015) ‘Using a matrix-analytical approach to synthesizing evidence solved incompatibility problem in the hierarchy of evidence’, Journal of Clinical Epidemiology. Elsevier Inc, 68(11), pp. 1251–1260." } ] }, { "id": "25901", "date": "04 Dec 2017", "name": "Helene Moustgaard", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe overall topic of this paper is important: Blinding facilitates unbiased trial results, but blinding is less used in surgical trials (and less frequently discussed) than in trials of pharmacological interventions. A prevalent preconception is that blinding is not possible, or not relevant, in surgical trials. The main strength of the paper is that it illustrates well this to be ill founded.\nHowever, we wondered whether the manuscript could be further improved. First, we were in doubt what the specific aim of the paper was. We assumed that the authors aimed to discuss the methodological role of binding in surgical trials (and not primarily to present and reflect on their previous review of surgical placebo trials)? If so, it would have strengthened the paper if it had provided some reflections on the barriers to the use of blinding in surgical trials, the ethical issues involved (and how we can best balance the need for reliable evidence for effects of surgery against the risks or discomforts patients may be exposed to in blinded surgical trials), as wells as discuss what measures could be taken to remedy the current situation where blinding is used infrequently in surgical trials. In general, the reader would be better prepared if the paper clearly stated whether the topic it addresses is blinding in surgical trials or use of placebo in surgical trials and whether the main focus is providing practical examples or providing a general overview of the topic. It would be helpful if the paper defined central terms used. For example, what is meant by “interventional procedures”? Does that include acupuncture, radiotherapy, and physiotherapy? Also, how do the authors conceptualize “subjective outcomes vs. objective outcomes” [1].\nThe central section Bias specific to surgical trials appears compressed. It would help the reader if the section could more clearly show how the example provided introduces bias, and in what way this bias is specific too surgery (rather than just more frequently encountered).\n\nOf minor importance was that we were puzzled by some of the references used. For instance, in the Introduction, there is a reference to a study by Wood et al [2], but several more recent studies have looked at this issue [3]. Also, in the section Blinding of outcome assessors the authors refer to a study by one of the undersigned, to the effect that the study documents a “Pygmalion effect” in un-blinded studies [4]. The term, however, was not used in the original publication. It would possibly help the reader if the authors referred to publications that provide a general overview of blinding in trials (and not only surgical trials).\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3338", "date": "30 Jan 2018", "name": "Karolina Wartolowska", "role": "Author Response", "response": "Our aim was to discuss the methodological role of blinding in surgical trials and to demonstrate that blinding can and should be done in surgical trials, especially, if the outcomes are subjective. The need to control for bias in surgical trials is often not recognised. We agree with the Reviewer that there is a preconception/assumption that blinding in surgical trials is not possible or not necessary. Surgery may mean an open surgery undertaken to preserve life. In such cases the outcome is objective, binary and with low risk of bias. However, nowadays, many invasive procedures are performed not to save lives but to improve the quality of life, function or to reduce pain. As a consequence, their outcomes are highly subjective, and as such, prone to bias. This problem seems to be unappreciated in clinical and research community. In this paper, we wanted to demonstrate that blinding can be achieved in surgical trials and that it is necessary if the outcomes are subjective. Placebo-controlled surgical trials represent a single type of trial that requires blinding, but similar arguments apply to trials comparing two different surgical methods. In this paper, we did not discuss the placebo because our main focus was blinding as a way to control bias. We used placebo-controlled surgical trials as an example but similar arguments apply to trials comparing two different surgical methods. We have already written about barriers in completing placebo-controlled surgical trials (Wartolowska et al., 2016), about the balance between harms and benefits of such trials (Wartolowska et al., 2014) and about the ethical implications of placebo in surgery (Savulescu, Wartolowska and Carr, 2016)(George et al., 2016). We have used acupuncture as an example of an experimental study on placebo effect because acupuncture is the only type of invasive procedure used in such studies. We have included radiofrequency procedures because they change anatomy. In this paper, we have defined surgery as “any interventional procedure that changes the anatomy and requires a skin incision or the use of endoscopic techniques; dental studies were excluded. We used the term placebo to refer to a surgical placebo, a sham surgery, or an imitation procedure intended to mimic the active intervention; including the scenario when a scope was inserted and nothing was done but patients were sedated or under general anaesthesia and could not distinguish whether or not they underwent the actual surgery”.(Wartolowska et al., 2014) We have added these definitions to the manuscript. “Outcomes were classified as “subjective”, i.e., patient-reported and depending on the patients’ perception and cooperation, “assessed”, i.e., subjective ratings by external assessors and depending upon their judgment, and “objective”, i.e., measured using devices or laboratory tests and independent of patients’ or observers’ perception, for example, weight.”(Wartolowska et al., 2016) As suggested by the Reviewers, we have explained in the section titled “Bias specific to surgical trials” that by including only patients with non-life-threatening symptoms or with sufficiently severe symptoms on the day of surgery a bias is created, which is difficult to control for. As suggested by the Reviewers, we have added the paper by Page et al. as a reference in this paragraph. We have also added a reference that provides a general overview of blinding in trials (Hróbjartsson and Boutron, 2011) and provided references to Rosenthal’s papers to explain the “Pygmalion effect”.(Rosenthal and Jacobson, 1968) REFERENCESGeorge, A. et al. (2016) ‘When should placebo surgery as a control in clinical trials be carried out?’, The Bulletin of the Royal College of Surgeons of England, 98(2), pp. 75–79.Hróbjartsson, A. and Boutron, I. (2011) ‘Blinding in randomized clinical trials: Imposed impartiality’, Clinical Pharmacology and Therapeutics. Nature Publishing Group, 90(5), pp. 732–736.Rosenthal, R. and Jacobson, L. (1968) ‘Pygmalion in the classroom’, The Urban Review, 3(1), pp. 16–20.Savulescu, J., Wartolowska, K. A. and Carr, A. (2016) ‘Randomised Placebo-Controlled Trials of Surgery: Ethical Analysis and Guidelines’, J Med Ethics, 42(12), pp. 776–783.Wartolowska, K. et al. (2014) ‘Use of placebo controls in the evaluation of surgery: systematic review.’, BMJ (Clinical research ed.), 348(May), p. g3253.Wartolowska, K. et al. (2016) ‘Feasibility of surgical randomised controlled trials with a placebo arm: a systematic review’, BMJ Open, 6(3), p. e010194.Wartolowska, K. A. et al. (2016) ‘The Magnitude and Temporal Changes of Response in the Placebo Arm of Surgical Randomized Controlled Trials - A Systematic Review and Meta-Analysis’, Trials. Trials, 17(1), p. 589." } ] } ]	1	https://f1000research.com/articles/6-1663
https://f1000research.com/articles/5-2744/v1	22 Nov 16	{ "type": "Research Article", "title": "Communicating behavioral genetics: Charting the limits of the genetic interpolation effect", "authors": [ "Alexandre Morin-Chassé" ], "abstract": "Background: Scientific research has linked genetic predispositions to complex social behaviors and orientations. Recent empirical work in science communication suggests that the dissemination of these research findings can impact on public beliefs about the influence of genetics. The genetic interpolation hypothesis posits that exposure to this type of news causes the public to update their views about the influence of genetics on human beings and to infer greater genetic causation for other social traits that are not mentioned in the news content. The main purpose of this study is to test whether the genetic interpolation effect also emerges following exposure to a soft treatment: a short summary paragraph rather than a real news article. It also tests if specifying that a particular gene has a marginal effect succeeds at moderating the genetic interpolation effect. Finally, the study tests a counter-hypothesis: instead of resulting from belief updating, the genetic interpolation effect is triggered by the simple act of thinking about genetics shortly before reporting beliefs. Methods: In total, 2080 respondents were recruited from a pre-existing online panel and were randomly split into four experimental groups: group 1) no message; group 2) a paragraph about how genes can impact on voting behavior; group 3) the same paragraph as group 2, plus an indication that the effect of a particular gene is small; group 4) a paragraph about how genetics impact on physical traits such as eye color. Subjects were then asked to evaluate the role of genetics in causing three traits: voting at election; intelligence; and natural hair style (curly or straight). Results: The analyses reveal no evidence supporting the genetic interpolation hypothesis, the moderation hypothesis or the counter-hypothesis. Conclusion: Overall, the results suggest that exposure to a paragraph describing how genes can impact on complex social behavior is not sufficient to trigger the genetic interpolation effect.", "keywords": [ "science communication", "public understanding of science", "behavior genetics", "genetic attribution", "biased assimilation", "genetic interpolation effect", "media effects", "survey experiment" ], "content": "Background\n\nThe field of behavioral genetics works at improving scientific understanding of human and animal social behaviors by investigating whether these behaviors are partly influenced by genetic predispositions. The causal framework that commonly underlies behavior genetics takes the form of a chain reaction, linking genes, at one end, to neural processes, mental abilities and temperament, personality traits, world views, attitudes, and behaviors1,2. Survey research and focus groups suggest that the public generally adheres to a belief system that is consistent with this causal framework3–5. Indeed, on average, people attribute a great role to genetics in explaining biological differences (i.e., eye color, height), a moderate influence for talents and social orientation (i.e., intelligence, sexual orientation), and a weak influence for complex social behavior (i.e., having debts, preferring Apple to Microsoft)6. However, the media sometimes presents research findings in a way that sharply contrasts with both the public understanding of genetics and actual research conclusions. Indeed, when paying attention to the language science reporters choose to describe research findings, it is often unclear whether the influence of genetics is deterministic, prominent, moderate, or marginal7,8.\n\nMany academics have expressed concern over the risk that the oversimplification of genetic causation in the mass media could lead the public to misinterpret research findings9–11. Recent empirical works in the field of science communication have offered initial support for this concern. It is not uncommon to see a news story reporting on the results of a scientific study suggesting that genetics can influence complex social behavior or orientation X. Experimental studies have tested how people react to this kind of news6. Two findings arise from this research. First, there is evidence of a persuasion effect: people exposed to this information increase the influence they attribute to genetics in explaining the characteristic X described in the article. However, the results also show that disseminating behavioral genetics leads some members of the public to infer greater genetic causation for other complex social traits, which were not the focus of the study presented. This side-effect has been called the genetic interpolation effect.\n\nIt is presumed that people infer greater genetic causation for other traits because they update their belief system about the overall role that genetics plays in defining social traits. The psychological mechanism at play here is likely the anchoring and adjustment heuristic12,13. This heuristic is a cognitive shortcut people often use in their everyday life to cope with uncertainty. Many individuals, even expert geneticists, would feel ambivalent if they had to assess the overall influence genetics has on a social orientation or a social behavior. Arguably, reading a news article presenting research findings from behavior genetics can offer a valuable piece of information to complete this task. Some people may react to behavior genetics by thinking to themselves that \"if genetics is strong enough to have a significant influence on this complex social trait, then its general influence on other social traits must be stronger than I had imagined,\" thus causing them to infer greater genetic influence for other complex characteristics not mentioned in the news article content.\n\nThe purpose of this study is threefold. First, the main goal is to test if the persuasion effect (H1) and the genetic interpolation effect (H2) also emerge following exposure to a softer treatment. Addressing this issue is necessary to better capture what kind of message can trigger the genetic interpolation effect. While previous studies exposed participants to real news articles, here subjects are exposed to one paragraph claiming that genes can influence a complex social behavior: voting or abstaining at elections. Second, the experiment was designed to test whether clarifying that a particular gene involved in causing this behavior only has a marginal effect and can moderate the size of the persuasion effect (H3). If this was found to be the case, one could expect it to result in much weaker belief updating for other non-related traits, thus contributing to moderating the size of the genetic interpolation effect as well (H4). Finally, this study was also designed to test a counter-hypothesis, an alternative explanation for the genetic interpolation effect. This alternative explanation posits that, instead of resulting from belief updating, the genetic interpolation effect is due to the simple fact that thinking about genetics shortly before reporting beliefs makes the genetic argument more accessible to participants’ minds (H5). If this was the case, being reminded that genetics can influence various physical traits – a “placebo” message – would suffice to move participants’ average response.\n\n\nMethods\n\nThis study was conducted as part of the Short Study Program of Time-Sharing Experiments in the Social Sciences. A web survey was fielded by the firm Government for Knowledge during the month of August 2013. A sample of 2080 respondents was recruited from a pre-existing panel, with a response rate of 62.6% (AAPOR RR3; see Data availability). No participants were excluded from this study. Participants were randomly assigned to one of four experimental conditions without knowing it. Table 1 reports the stimulus for each experimental condition. Table 2 reports a randomization check.\n\nFollowing treatment exposure, subjects were asked how much they believe genetics (as opposed to the environment) impacts on three features: “turning out to vote,” “intelligence,” and “natural hairstyle (curly or straight).” Items were presented in random order. Table 3 shows the eleven-point response scale. We report all measures and manipulations.\n\nThe statistical analyses were performed using Stata 14. H1, H2 and H5 require testing whether the mean genetic attribution for a trait is higher in a specific treatment group compared to the control group. We test these hypotheses by running various Welch’s t-tests, which, contrary to Student’s t-tests, do not assume that the two groups under comparison have equal variances. Furthermore, H3 and H4 predict moderation effects. These are tested by fitting OLS regression models that predict the genetic attribution for the relevant trait with three dummy variables, one for each group. We then use the functions margins, dydx and lincom to compare coefficient values and perform difference in difference tests (statistical threshold, p<0.05 two-tailed).\n\n\nResults\n\nTable 4 presents the results of the experiment. The first element worth noticing is that the mean genetic attribution for turnout in the control group is much higher than what one would intuitively expect, with a mean of 27.24 on a scale ranging from 0 to 100. We ran Welch t-tests to assess the impact of treatment exposure. The second column of Table 3 presents the results of comparisons between the group exposed to genopolitics (Group 2) and the control group (Group 1). The results for the turning out to vote item show evidence of a statistically significant, but substantially small persuasion effect, offering support for H1. However, although the average genetic attribution for intelligence in Group 1 is higher than in the control group, this difference is too small to reach statistical significance. Therefore, we must reject H2.\n\nNote: p<0.05; *p<0.01. Two-tailed tests.\n\nResults presented in the third column indicate that there is still a significant persuasion effect among the group exposed to the paragraph insisting on the marginal effect of a particular gene (Group 3). The fourth column presents the result of a difference in difference comparison, testing whether adding this word of caution about the marginal influence of a specific gene succeeds at moderating the persuasion effect. The results displayed in the first row of this column indicate no significant difference between the persuasion effects caused by these two paragraphs about genopolitics, thus leading us to reject H3. Considering that both H2 and H3 are rejected, it should be no surprise to find that the second row of the fourth column offers no support for H4 either. Finally, the fifth column of Table 3 suggests that, contrary to what H5 predicts, presenting a paragraph describing how genetics impacts on human physical traits is not sufficient to generate the genetic interpolation effect.\n\nNoticeably, the results also present an unanticipated finding: compared to Group 1, Group 2 and Group 3 show lower average genetic attribution for natural hair style. The genetic interpolation hypothesis offers no explanation for this phenomenon, but something else may be at play here.\n\nSurvey satisficing occurs when respondents use strategies and cognitive shortcuts to offer a survey response without making all the efforts to ensure that the answer they give best corresponds to their opinion, their point of view or, as it is the case here, their belief14. Strength-lining is a form of satisficing that consists of picking the same answer repeatedly for different questions without differentiating between them15. A closer look at the response distribution offers reasons to believe that exposure to either of the two genopolitics treatment paragraphs demotivated some participants to offer honest answers. Indeed, in both of these groups, approximately 10% of respondents picked the same response option for our three question items; in comparison, the equivalent figures for the control group and the group exposed to the placebo treatment are 6.45% and 6.65% (pairwise differences are statistically significant at p<0.05). But crucially, most straightliners answered the middle option category (50% genetics and 50% environment) for all three questions.\n\nGreater satisficing thus pulls the average genetic attribution response further toward the center of the scale. This unexpected finding may partly explain the surprising negative effect observed for the hair style response item. Admittedly though, it might as well partly account for the positive effect observed on the voting at election item, a positive effect that what would otherwise be assimilated to the persuasion effect.\n\n\nDiscussion\n\nThe main purpose of this study was to test whether exposure to a paragraph on behavior genetics can generate the genetic interpolation effect. The experimental evidence presented above offers no indication that this is the case. However, both paragraphs presenting behavior genetics findings were successful at moving reported beliefs about how much genetics impacts on the trait presented in the paragraph – here, voting at elections. Yet, a closer look at the response distribution suggests that we should not overemphasize this finding, for a part of it may be attributable to lower response quality. Furthermore, our results indicate that insisting on the small role of a particular gene fails at moderating this persuasion effect. Finally, the results suggest that making genetic causation more accessible to the mind using a placebo message does not suffice to produce the persuasion or the genetic interpolation effect.\n\nNevertheless, the key finding here is related to the fact that the genetic interpolation effect was not exhibited. One likely explanation for this relates to the small size of the persuasion effect, more or less 4 percentage points. For the sake of comparison, in previous experiments, participants exposed to a whole news article about genopolitics showed persuasion effects ranging between 10 and 15 percentage points6. With this in mind, it seems plausible to interpret this null finding as an indication that a short paragraph on behavior genetics is a treatment condition that is not strong enough to cause people to update their general belief framework about the influence of genetics.\n\nAdditional research is needed to clarify the reasons why neither the moderation hypothesis nor the counter-hypothesis were confirmed. Indeed, it remains unclear whether these hypotheses would be supported if subjects were exposed to a real, and presumably more credible, news article instead. In spite of its null finding, the present study will hopefully help orient future research investigating how people react to behavior genetics. Indeed, our results help to chart the limits of the genetic interpolation effect by showing that not every type of message about behavior genetics succeeds in triggering this side-effect.\n\n\nEthical approval\n\nThe study was approved by the Comité d’éthique de la recherche en arts et en sciences of the Université de Montréal.\n\n\nData availability\n\nThe dataset and the pollster report for this study are hosted on the Open Science Framework: DOI: 10.17605/OSF.IO/2UBP216.", "appendix": "Author contributions\n\n\n\nAMC conducted all the work related to this study.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nData collected by Time-sharing Experiments for the Social Sciences, NSF Grant 0818839, Jeremy Freese and James Druckman, Principal Investigators. The author also benefited from a Ph.D. Scholarship awarded by the Fonds de recherche du Québec - Société et culture at the time the survey was fielded.\n\n\nAcknowledgments\n\nThe author is grateful to the Center for Research on Ethical, Legal and Social Implications of Psychiatric, Neurologic, and Behavioral Genetics, based at Columbia University, for offering a stimulating research environment during his stay as a postdoctoral researcher.\n\n\nReferences\n\nDawes C, Cesarini D, Fowler JH, et al.: The relationship between genes, psychological traits, and political participation. American Journal of Political Science. 2014; 58(4): 888–903. Publisher Full Text\n\nMondak JJ, Hibbing MV, Canache D, et al.: Personality and civic engagement: An integrative framework for the study of trait effects on political behavior. American Political Science Review. 2010; 104(1): 85–110. Publisher Full Text\n\nMORI survey of the People’s Panel: Public attitudes to human genetic information. Technical report, Human Genetics Commission, 2001. Reference Source\n\nShostak S, Freese J, Link BG, et al.: The politics of the gene: Social status and beliefs about genetics for individual outcomes. Soc Psychol Q. 2009; 72(1): 77–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nParrott RL, Silk KJ, Condit C: Diversity in lay perceptions of the sources of human traits: genes, environments, and personal behaviors. Soc Sci Med. 2003; 56(5): 1099–1109. PubMed Abstract \| Publisher Full Text\n\nMorin-Chassé A: Public (mis)understanding of news about behavioral genetics research: A survey experiment. BioScience. 2014; 64(12): 1170–1177. Publisher Full Text\n\nCappella JN, Mittermaier DJ, Weiner J, et al.: Framing genetic risk in print and broadcast news: A content analysis. In 93rd Annual meeting of the National Communication Association. Chicago IL. Reference Source\n\nBrechman J, Lee CJ, Cappella JN: Lost in translation? a comparison of cancer-genetics reporting in the press release and its subsequent coverage in lay press. Sci Commun. 2009; 30(4): 453–474. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNelkin D, Lindee SM: The DNA Mystique: The Gene as a Cultural Icon. WH Freeman and Co, 1995. Reference Source\n\nPeters T: Playing God?: Genetic Determinism and Human Freedom. Routledge, 2014. Reference Source\n\nKrimsky S, Gruber J: Genetic Explanations: Sense and Nonsense. Harvard University Press, 2013. Reference Source\n\nTversky A, Kahneman D: Judgment under uncertainty: Heuristics and biases. In Dirk Wendt and Charles Vlek, editors, Utility, Probability, and Human Decision Making. D. Reidel Publishing Company, 1975; 11: 141–162. Publisher Full Text\n\nFurnham A, Boo HC: A literature review of the anchoring effect. J Socio Econ. 2011; 40(1): 35–42. Publisher Full Text\n\nKrosnick JA: Response strategies for coping with the cognitive demands of attitude measures in surveys. Appl Cogn Psychol. 1991; 5(3): 213–236. Publisher Full Text\n\nKaminska O, McCutcheon AL, Billiet J: Satisficing among reluctant respondents in a cross-national context. Public Opin Q. 2010; 74(5): 956–984. Publisher Full Text\n\nMorin-Chassé A: Communicating behavior genetics: Charting the limits of the genetic interpolation effect. Open Science Framework, 2016. Data Source" }	[ { "id": "18117", "date": "08 Dec 2016", "name": "Brian M. Donovan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn “Communicating behavioral genetics: Charting the limits of the genetic interpolation effect” the authors report research from an experimental trial run online which explores how exposure to genetic information about human behaviors affects beliefs about the genetic (versus environmental) determinants of social behavior. This topic is an important area for research given the often widespread misunderstanding of genetics and behavior, which is both perpetuated through genotype in the media and also through science education. Furthermore, genetic determinism has been shown to be implicated in a variety of social prejudices by social psychologists. Consequently, the null findings reported in this research could be significant to many science communication scholars, science education researchers, or social psychologists interested in public understanding of genetics. To make that contribution more clear, however, the author needs to attend to some theoretical and methodological issues in this present draft of the manuscript.\n\nThose issues are:\nA clearer and stronger theoretical and/or empirical rationale for testing a softer treatment to chart the limits of the genetic interpolation effect.\n\nClearly defined confirmatory and exploratory hypothesis tests which are then mapped onto a post-hoc power analysis to demonstrate that the trial was well-powered, and thus, that the null findings are significant.\n\nMore care needs to be taken in interpreting the null findings. At present, the authors provide a partial explanation for the null findings with the idea of straightlining. However, by their own account this explanation might only capture 6-10% of the null effect.Thus, a more convincing explanation for the null effect is needed to guide future research.\n\nIn sum, the significance of a null-experimental finding should be judged on its ability to guide future research. Null findings, when well powered, should help researchers decide which paths NOT to go down. And, they should provide some theoretical reason for why such paths lead nowhere. At present, the manuscript does not accomplish this task. But, with major revisions it might be able to make this contribution. Thus, I recommend that the study be approved with reservations. If all of the issues outlined below in my critique of the piece can be addressed by the author, then I think this piece will make a significant contribution to the public understanding of genetics.\n\nTheoretical issues:\n\nI would have liked to see a more nuanced discussion about the possible mechanisms which link the reading of behavioral genetics research to genetic attributions of complex human traits. For example, if the genetic interpolation effect is actually based on anchoring and adjustment, then one would expect that individual differences in people’s prior conceptions about the genetic basis of human traits might moderate the activity of the genetic interpolation effect.\n\nFor example, the author hypothesizes that “Some people may react to behavior genetics by thinking to themselves that \"if genetics is strong enough to have a significant influence on this complex social trait, then its general influence on other social traits must be stronger than I had imagined,\" thus causing them to infer greater genetic influence for other complex characteristics not mentioned in the news article content.”\n\nBut, what if people already strongly believe that social traits are caused by genes. In this situation, a ceiling effect might exist, thus preventing the statistical detection of the genetic interpolation effect. Under a ceiling effect model, we would expect a Treatment x Prior genetic belief interaction, where those who do not attribute social traits to genes before reading are affected by the experiment and those who do are not impacted by treatment. Conversely, it could also be the case that those who are generally averse to the idea that genes cause human social traits are inoculated against any impact that reading about behavioral genetics might have on genetic interpolation. Thus, the Treatment x Prior genetic belief interaction on the genetic interpolation effect could be the opposite of what I have previously stated.\n\nThe point is that prior knowledge and beliefs could have washed out the treatment effects and produced the null findings of this experiment. And, there is a large body of reading comprehension research which shows that the meaning people construct from readings varies with their prior knowledge. Hence, to interpret these null findings some discussion of the impact of prior knowledge is needed both in the theoretical motivation for the study and during the discussion. Indeed, the authors allude to this need when they discuss an unexpected finding in their results. They state:\n\n“Noticeably, the results also present an unanticipated finding: compared to Group 1, Group 2 and Group 3 show lower average genetic attribution for natural hair style. The genetic interpolation hypothesis offers no explanation for this phenomenon, but something else may be at play here.”\n\nPerhaps the explanation is that individual differences in people’s prior beliefs caused the null effect? The authors partly explain this finding with straightliners who varied significantly in proportion by condition. However, only 6-10% of straightliners could be inferred from their responses in each condition. So, the above issue – the issue of prior knowledge and beliefs affecting how people constructed meaning from the texts – might help to explain the other part. I would encourage the authors to analyze and report any findings they have (or have not reported) which explore how responses in the baseline data help to resolve the mechanism for the null results.\n\nMethodological issues:\n\nBecause the researchers apparently did not measure prior knowledge of genetics or genetic beliefs prior to reading, then this could also be discussed as a methodological limitation of the present experiment.\n\nThe theoretical and empirical rationale for testing a shorter treatment given in the introduction is not convincing nor is it compelling. For example, the authors state, “The purpose of this study is threefold. First, the main goal is to test if the persuasion effect (H1) and the genetic interpolation effect (H2) also emerge following exposure to a softer treatment. Addressing this issue is necessary to better capture what kind of message can trigger the genetic interpolation effect”. If this study is telling us anything about the world, then it should tell us what it is actually modeling in the world through the experimental design. For example, is the shorter reading on behavioral genetics more akin to what science students might read in a textbook, and thus, worth investigating? Or, is it the kind of message that people might come across on PubMed? In short, why is the shorter reading important to investigate?\n\nThere are four conditions and 2080 participants. Given that the main finding in this study is a null finding, some care and effort needs to be taken to explain the a priori power analysis and post-hoc power analysis for the trial. For example, if the experiment is underpowered, then the significance of the null finding is diminished if not destroyed.\n\nThis issue is exacerbated by the multiple comparisons in the study. With four groups in the study there are three comparisons that can be made with the control group. Then, there are three different measures. So, in sum total there are at least nine-different statistical tests that could be run to test hypotheses. The authors should outline which of these tests are the confirmatory tests and which are the exploratory tests. Alpha values, and hence p-values, should be adjusted to account for Type-I error on the confirmatory tests. And, then, based on the actual effect sizes, and the adjusted alpha criterion, the authors should report what their post-hoc power was on each test. If the reach 80% power and their finding is still null, then such a null finding, in my mind, is worthy of indexing. However, if the authors were underpowered, then the contribution of these findings to the research community is less clear.\n\nIf we take the authors at their word, then with 2080 people in their panel and a response rate of 62.6%, there are about 1300 people enrolled in the experiment. If we divide that number in half (n = 650), that is the amount of people present in any single comparison with the control. Then, we can divide 0.05/3 to account for the three different variables per treatment-control contrast. If we assume there is no baseline data which would improve the precision of the estimate by increasing the R2 in the models, then any single treatment-control contrast could detect an effect size of d = 0.25 or greater. If we, instead, adjust alpha to account for all possible treatment control contrasts on all variables (i.e. 9 tests or 0.05/9 = 0.0055), then effects of d = 0.28 or greater should be detectable. So, the question is, how big were the effects. Unfortunately, the standard deviation for the control group is not reported, nor are the pairwise effect sizes. If the authors provided that information, then the readers would have more confidence that the null effects are significant and merit indexing.", "responses": [ { "c_id": "2356", "date": "08 Dec 2016", "name": "Alexandre Morin-Chassé", "role": "Author Response", "response": "M. Donovan, thanks for your careful reading of this paper. Your suggestions and critiques will help get the most out of this study. I will wait until I receive additional reviews and devote some time in January to work on writing a revised version which will address all of your concerns." } ] }, { "id": "17905", "date": "10 Jan 2017", "name": "Kostas Kampourakis", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a review of the manuscript titled \"Communicating behavioral genetics: Charting the limits of the genetic interpolation effect\". I have strong reservations about the manuscript on various grounds, and I hope that the author will manage to revise it.\nFirst of all, the author needs to justify why we should care at all about what he calls \"the genetic interpolation effect\". With this, I do not mean to suggest that we should not care. Rather, I suggest that the author needs to make a stronger case for this. The author writes that \"Experimental studies have tested how people react to this kind of news\", but the author only cites his own previous work. Are these author's studies all the experimental studies on this topic? Or are there others that have not been mentioned? Is this effect well-established in research? Or is it something recently \"discovered\"?\n\nThe author writes that \"This side-effect has been called the genetic interpolation effect.\" By whom? Why? How strong this effect is? How prevalent? No references are given and so we have no idea about these. I am also wondering about the name itself. The author writes that \"However, the results also show that disseminating behavioral genetics leads some members of the public to infer greater genetic causation for other complex social traits, which were not the focus of the study presented.\" This looks more like extrapolation to me. Perhaps the author is right, and my point is not to question the choice of the name. Rather my point is to suggest that we need more information about this effect, which is the focus of the article.\n\nThere are studies by Ilan Dar Nimrod (http://sydney.edu.au/science/people/ilan.dar-nimrod.php) that I find very relevant and that should have at least been discussed in this article. Celeste Condit has also conducted a few studies on how people interpret news articles on genetics. Both of these scholars would be appropriate reviewers for this article. My suggestion would be for the author to briefly review the findings of their research, and relate his own research to those in order to make more explicit what his contribution is. Individual studies may make interesting contributions, but generalizations are not easy to make even if the sample is large.\n\nSome more information about the anchoring and adjustment heuristic would be useful. These are not sufficiently described in the current article, and so one must read the cited articles in order to understand what it is about and how it relates to the research presented therein.\n\nThere are some problems with the hypotheses made by the author. Does H2 presuppose H1? This is not clear. My understanding is that it does, i.e. that people first have to increase the influence they attribute to genetics in explaining a characteristic after reading the article, in order to subsequently infer greater genetic causation for other complex social traits. This is at least the inference I make from author's description of H3 and H4. It is of course plausible that this is not the assumption made by the author. But then the author should explain why this is the case. Even if I am wrong, the author should ensure that readers will not make the same mistake I have made.\nCommenting on the statistical methods used falls outside my modest area of expertise. Therefore, I will only comment on the conclusions made assuming that the statistical analysis is appropriate.\n\nThe author writes: \" A sample of 2080 respondents was recruited from a pre-existing panel, with a response rate of 62.6%\" Does this mean that approximately 1302 people responded? If yes, why isn't this made clear so that we know what the sample of the study was? That 38% of those asked declined to respond may be interesting for those conducting this kind of surveys. However, in this case we only need to know the actual sample size; how many people were asked to participate is of secondary importance.\n\nI am wondering how the reliability and the validity of the inferences made from participants responses was established. Did the author ask experts to read the stimuli and confirm that they are appropriate to measure what the author intends to measure? Were these stimuli used in a pilot study of some kind before this study? For instance the author writes that (Table 1) “Between 1993 and 2003, scientists from around the world have worked on coding the chemical components of human DNA.\" The expression \"coding the chemical components\" makes no sense to me. What happened during the HGP was that researchers developed methods to find the sequence of the chemical components (bases A,T,C,G in nucleotides) of the human genome. Decoding this sequence, i.e. finding the information encoded therein, is something that they are still working on. The expression \"coding the chemical components\" could be very misleading and I do not know if this was done in purpose or accidentally.\n\nOn the same topic, I do not see why the author would expect that H5 would be confirmed. The author concludes that \"Finally, the fifth column of Table 3 suggests that, contrary to what H5 predicts, presenting a paragraph describing how genetics impacts on human physical traits is not sufficient to generate the genetic interpolation effect.\" There are numerous studies that provide evidence that people intuitively tend to think about biological characteristics as influenced, if not determined, by genes. People tend to take for granted that \"physical traits\" \"are strongly influenced by genetics\". Confusion begins when people mistakenly start wondering how much of a trait is due to genes or due to environment, as they often fail to understand that it is the interaction of the two that brings about a phenotypic outcome and thus they cannot be separated. Now, it seems to me that what the author is testing here is another extrapolation effect: whether a statement about biological traits would form the basis for inferences for a behavioural trait. This is interesting but seems to me to be a different effect than the one that the author wants to test, rather than \"an alternative explanation for the genetic interpolation effect.\"\nBased on all this, I suggest that the author carefully reconsiders his conclusions. I am not at all convinced that this conclusion is valid: \"it seems plausible to interpret this null finding as an indication that a short paragraph on behavior genetics is a treatment condition that is not strong enough to cause people to update their general belief framework about the influence of genetics.\" I am inclined to think that when it comes to genetics even a replacement effect is possible, i.e. reading a highly deterministic headline could make people think in more deterministic terms about genetics. There is no research on that, but some relevant research suggests that this could be possible (e.g. Ecker , U. K. , Lewandowsky , S. , Chang , E. P. , & Pillai , R. ( 2014 ). The effects of subtle misinformation in news headlines. Journal of Experimental Psychology: Applied,20 ( 4 ), 323 – 335.). This is just speculation based on personal experience, and we need research to draw conclusions. The conclusions of this article seem to point to the opposite direction, but I am not sure about their validity. The author should work more and reconceptualize the available data in order to be more convincing that his conclusions are valid.\nTherefore, I have strong reservations about the quality and the conceptual foundations of this study, and I would like to see if the author will manage to address my concerns.", "responses": [] } ]	1	https://f1000research.com/articles/5-2744
https://f1000research.com/articles/7-112/v1	25 Jan 18	{ "type": "Research Article", "title": "Effect of maize root exudates on indole-3-acetic acid production by rice endophytic bacteria under influence of L-tryptophan", "authors": [ "Arun Karnwal", "Aradhana Dohroo", "Aradhana Dohroo" ], "abstract": "Background: It is assumed that plant growth regulators produced by beneficial bacterial species could also influence plant growth. IAA is a major plant growth regulator responsible for stimulation of plant growth. There are several microorganisms which are naturally responsible for L- tryptophan metabolism. Methods: In total, 56 indigenous morphologically distinct isolates from rice roots were selected and subsequently characterized with biochemical tests, 16S rRNA sequencing and plant growth promoting activities. Pseudomonas fluorescens RE1 (GenBank: MF102882.1) and RE17 (GenBank: MF103672.1) endophytes resulted in better PGP activity against the other 54 isolates. Both endophytes were tested to screen indole-3-acetic acid production ability in pure culture conditions with L-tryptophan at 0, 50, 100, 200 and 500µg/ml concentrations. Results: P. fluorescens RE1 was recorded efficient for indole production in comparison to P. fluorescens RE17 at various L-tryptophan concentrations. P. fluorescens RE1 was shown to produce between 0.8 µg/ml and 11.5µg/ml of indole at various tryptophan concentrations, while RE17 produced between 1.2µg/ml and 10.2µg/ml. At 200 and 500µg/ml tryptophan concentration, P. fluorescens RE17 produced 7.4pmol/ml and 9.3pmol/ml IAA, respectively. Conclusions: Inoculation of maize seed with P. fluorescens RE1 and RE17 showed a significantly higher level of IAA production in comparison to non-inoculated seeds. Current study outcomes proved that plant growth regulators produced by Pseudomonas species could also play a critical role in plant growth promotion.", "keywords": [ "Pseudomonas fluorescens", "endophytes", "maize", "rice", "tryptophan" ], "content": "Introduction\n\nIndole-3-acetic acid (IAA) is one of the most physiologically active phytohormones, and is a product of L-tryptophan metabolism (Yu et al., 2017). 80% of rhizospheric microorganisms naturally yield auxins as secondary metabolites due to the rich supplies of root exudates (Myresiotis et al., 2015). The rhizosphere is a highly selective area for host-microbe interaction, and during their life span few microbes may enter inside the plant tissue and stay without causing any negative symptoms (Santoyo et al., 2016). These indigenous colonizers reside in almost all internal tissues/cells of plant ranging from tissues of the roots to stem, leaf, flower, fruit and seed (Souza et al., 2015).\n\nThese endophytes actively or passively facilitate modification of morphology in the plant cell. It is reported by many workers that endophytes promote growth due to favourable adaptations to abiotic or biotic stresses (Nutaratat et al., 2014). Indigenous bacteria also promote plant growth through various mechanisms. These include direct and indirect mechanisms. Direct mechanisms involve various plant growth promoting hormones (i.e. indole-3-acetic acid, gibberelic acid, adenine-type cytokinins, phenylurea-type cytokinins, ethylene, and abscisic acid), solubilizing inorganic phosphate and atmospheric N2 fixation. However, indirect mechanisms involve production of various antimicrobial chemical, siderophores and lytic enzymes against the plant pathogens (Zhang et al., 2013). The aim of the present study is to analyse the effect of L-tryptophan and maize root exudates on the production of IAA by rice endophytic bacteria.\n\n\nMethods\n\nA total of forty healthy rice (Oryza sativa L. basmati) plants were randomly selected and collected from agricultural land situated at Dehradun (30° 19’ N, 78° 04’ E) Uttarakhand, India. Surface-sterilized roots were dissected into small pieces and 1g fresh root tissue was ground in sterile mortar and pestle with 0.85% sterilized saline solution. The ground tissue extract was serially diluted (sevenfold) in sterile saline and 100 µL aliquots were spread on nutrient agar plates (Hi-Media, India). Biochemical characterization of 56 endophytic isolates was carried out as described in Bergey’s manual of determinative bacteriology (Holt et al., 1994). On the basis of higher indole production ability, two isolates RE1 and RE17 were selected for further study and 16S rRNA analysis.\n\n16S rRNA sequencing and phylogenetic analysis were done for both isolates RE1 and RE17. Universal 16S rRNA primers (8F 5’ AGAGTTTGATCCTGGCTCAG 3’ and U1517R 5’ ACGG(A/C)TACCTTGTTACGACTT 3’) were used for 16S rRNA amplification of bacterial isolates under PCR reaction (Puente et al., 2004). The ProbeBase online utility was used to check primers specificity and the BLAST search facility at the NCBI (Genbank database). Multiple sequence alignment was performed by using MUSCLE alignment algorithm and PhyML software was applied for phylogenetic analysis (Dereeper et al., 2008).\n\nIsolate RE1 and RE17 were tested for indole production by using the method described by Karnwal (2009). Indole production was analyzed by mixing 4ml of Salkwaski’s reagent in 1ml of cell free filtrate. This mixture was incubated at 28 ±2°C for 15 min to observe pink coloration as positive indication of IAA production. Absorbance was measured at 535 nm using UV-VIS Spectrophotometer 2201 (Systronics, India). Standard curve of IAA was used to quantify indole production by bacterial isolates (Karnwal, 2009). ELISA (Phytodetek, Agdia Inc, Elkhart, IN, USA) was used to estimate IAA produced by RE1 and RE17 as described by Karnwal (2009).\n\nMaize (Zea mays L. Kissan) seeds were procured from the local market of Dehradun (30° 19’ N, 78° 04’ E) Uttarakhand, India. Surface sterilized seeds were soaked in 10 ml of bacterial suspension, grown in half strength tryptic soy broth (TSB), until 108 cell density was achieved after gentle agitation for 10–15 min. Pre-sterilized growth pouches supplemented with 30ml ml of sterile half-strength N-free Hoagland’s nutrient solution were inoculated with bacterial treated seeds aseptically (3 seeds per pouch and 3 pouches per treatment). Seeds treated with 0.1 M MgSO4 were considered as controls.\n\nBacteria layered maize seeds were inoculated in growth pouches and cultivated inside plant growth chambers at 100rpm. IAA concentration from the growth pouch supernatant was determined with ELISA (Phytodetek, Agdia Inc, Elkhart, IN, USA). Stock solutions of the IAA (10 µmole/ml) were prepared within absolute methanol and standard concentrations 78–2500 pmoles/ml (IAA) were used for standard curve preparation.\n\n\nResults\n\nIn the present study, 5.6 × 101 CFU/ml morphologically unambiguous indigenous bacteria were purified and selected for further analysis for phytohormone IAA production ability. Isolates RE1 and RE17 were identified on the basis of Gram stain, biochemical activities and sugar fermentation, as described in Bergey’s manual of determinative bacteriology (Holt et al., 1994) (Table 1). BLAST analysis of the 16S rRNA gene sequence of RE1 and RE17 isolates demonstrated maximum sequence similarity with the P. fluorescens strain ATCC 13525 (99%, Genbank Sequence ID: NR_114476.1) and P. fluorescens strain CCM 2115 (98%, Genbank Sequence ID: NR_115715.1), respectively as shown in phylogenetic tree analysis by using MUSCLE algorithm and PhyML phylogenetic tree creation software (Figure 1).\n\n(A) BLAST similarity search results and phylogenetic tree for isolate RE1; (B) Phylogenetic tree for isolate RE17.\n\nIn the deficiency of L-tryptophan, strain RE17 released considerable levels of indole (0.7µg/.ml) in comparison with RE1 (0.2µg/ml). In the presence of 50µg/ml of L-tryptophan, RE17 produced significantly higher concentrations of indole compared to RE1 (Figure 2). When 200µg/ml of L-tryptophan was added to the medium, RE1 and RE17 produced eight, and three times the concentration of indole produced at 50µg/ml of L-tryptophan concentration, respectively (Figure 2). It has been observed that RE17 has greater IAA production ability than RE1 (Figure 3). Varying levels of IAA were recorded with different concentrations of tryptophan, i.e. 0, 100, 200 and 500 μg/ml by RE1 as shown in Figure 3.\n\nThe concentrations of IAA secreted with maize root exudates in growth chamber studies with RE1 and RE17 were significantly higher (2.8 pmol/ml and 3.4 pmol/ml) than that of the control (0.2 pmol/ml), as shown in Figure 4.\n\n\nDiscussion\n\nIndigenous microbes colonize internal regions of the plant and are present in almost every plant globally (Ji et al., 2010). IAA secretion through endophytes is a beneficial trait leading to plant development (Ahmad et al., 2008). Therefore, current studies examine IAA generating endophytic P. fluorescens strains from rice roots, with many reporting the active role of tryptophan in the production of IAA by plant growth promoting bacteria (Karnwal, 2017; Stachecki et al., 2004). Tryptophan improves IAA biosynthesis in P. fluorescens strains associated with current research, revealing that it might be the precursor for IAA biosynthesis in these bacterial strains (Khan & Bano, 2016). It has been shown that L-tryptophan concentration affects biosynthesis of IAA at significant levels (Ignatova et al., 2015), however, in this study IAA production was between 1.3 and 9.3pmol/ml in both P. fluorescens strains used, with or without tryptophan. Using ELISA based studies, it was revealed that strain RE17 was the best IAA producer in the presence of maize root exudates in growth chamber study.\n\nThe rhizosphere is a rich environment for growth of microorganisms, and it generates a great diversity of microbes (Balseiro-Romero et al., 2016; Kierul et al., 2015; Vaishnav et al., 2016). The growth chamber study confirmed the beneficial potential of maize root exudates on IAA secretion. This supports the fact that plant roots produce some chemical substances (root exudates) that support the growth of rhizospheric microorganisms and their colonization (Kierul et al., 2015; Pereira & Castro, 2014). Hence, these strains have a potential of being developed as bio-inoculants.\n\n\nData availability\n\nBacterial isolate sequence data at NCBI:\n\nPseudomonas fluorescens strain RE1 16S ribosomal RNA gene, partial sequence: GenBank: MF102882.1.\n\nPseudomonas fluorescens strain RE17 16S ribosomal RNA gene, partial sequence: GenBank: MF103672.1.\n\nRaw data for biochemical analyses and IAA production: http://doi.org/10.17605/OSF.IO/X5Q46 (Karnwal, 2018).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAhmad F, Ahmad I, Khan MS: Screening of free-living rhizospheric bacteria for their multiple plant growth promoting activities. Microbiol Res. 2008; 163(2): 173–181. PubMed Abstract \| Publisher Full Text\n\nBalseiro-Romero M, Gkorezis P, Kidd PS, et al.: Enhanced Degradation of Diesel in the Rhizosphere of after Inoculation with Diesel-Degrading and Plant Growth-Promoting Bacterial Strains. J Environ Qual. 2016; 45(3): 924–932. PubMed Abstract \| Publisher Full Text\n\nDereeper A, Guignon V, Blanc G, et al.: Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res. 2008; 36(Web Server issue): W465–9. Epub 2008 Apr 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolt JG, Krieg NR, Sneath PH, et al.: Bergey’s Manual of Determinative Bacteriology. Baltimore: Williams & Wilkins, 1994. Reference Source\n\nIgnatova LV, Brazhnikova YV, Berzhanova RZ, et al.: Plant growth-promoting and antifungal activity of yeasts from dark chestnut soil. Microbiol Res. 2015; 175: 78–83. PubMed Abstract \| Publisher Full Text\n\nJi X, Lu G, Gai Y, et al.: Colonization of Morus alba L. by the plant-growth-promoting and antagonistic bacterium Burkholderia cepacia strain Lu10-1. BMC Microbiol. 2010; 10: 243. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKarnwal A: Production of indole acetic acid by fluorescent pseudomonas in the presence of L-tryptophan and rice root exudates. J Plant Pathol. 2009; 19(1): 61–63.\n\nKarnwal A: Isolation and identification of plant growth promoting rhizobacteria from maize (Zea mays L.) rhizosphere and their plant growth promoting effect on rice (Oryza sativa. L.). J Plant Prot Res. 2017; 57(2): 144–151. Publisher Full Text\n\nKarnwal A: F1000research DATA. 2018. Data Source\n\nKhan N, Bano A: Role of plant growth promoting rhizobacteria and Ag-nano particle in the bioremediation of heavy metals and maize growth under municipal wastewater irrigation. Int J Phytoremediation. 2016; 18(3): 211–221. PubMed Abstract \| Publisher Full Text\n\nKierul K, Voigt B, Albrecht D, et al.: Influence of root exudates on the extracellular proteome of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42. Microbiol. 2015; 161(1): 131–147. PubMed Abstract \| Publisher Full Text\n\nMyresiotis CK, Vryzas Z, Papadopoulou-Mourkidou E: Effect of specific plant-growth-promoting rhizobacteria (PGPR) on growth and uptake of neonicotinoid insecticide thiamethoxam in corn (Zea mays L.) seedlings. Pest Manag Sci. 2015; 71(9): 1258–1266. PubMed Abstract \| Publisher Full Text\n\nNutaratat P, Srisuk N, Arunrattiyakorn P, et al.: Plant growth-promoting traits of epiphytic and endophytic yeasts isolated from rice and sugar cane leaves in Thailand. Fungal Biol. 2014; 118(8): 683–694. PubMed Abstract \| Publisher Full Text\n\nPereira SI, Castro PM: Diversity and characterization of culturable bacterial endophytes from Zea mays and their potential as plant growth-promoting agents in metal-degraded soils. Environ Sci Pollut Res. 2014; 21(24): 14110–14123. PubMed Abstract \| Publisher Full Text\n\nPuente ME, Bashan Y, Li CY, et al.: Microbial populations and activities in the rhizoplane of rock-weathering desert plants. I. Root colonization and weathering of igneous rocks. Plant Biol (Stuttg). 2004; 6(5): 629–642. PubMed Abstract \| Publisher Full Text\n\nSantoyo G, Moreno-Hagelsieb G, Orozco-Mosqueda Mdel C, et al.: Plant growth-promoting bacterial endophytes. Microbiol Res. 2016; 183: 92–99. PubMed Abstract \| Publisher Full Text\n\nSouza Rd, Ambrosini A, Passaglia LM: Plant growth-promoting bacteria as inoculants in agricultural soils. Genet Mol Biol. 2015; 38(4): 401–419. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStachecki S, Praczyk T, Adamczewski K: Adjuvant effects on plant growth regulators in winter wheat. J Plant Prot Res. 2004; 44(4): 365–371. Reference Source\n\nVaishnav A, Kumari S, Jain S, et al.: PGPR-mediated expression of salt tolerance gene in soybean through volatiles under sodium nitroprusside. J Basic Microbiol. 2016; 56(11): 1274–1288. PubMed Abstract \| Publisher Full Text\n\nYu X, Li Y, Cui Y, et al.: An indoleacetic acid-producing Ochrobactrum sp. MGJ11 counteracts cadmium effect on soybean by promoting plant growth. J Appl Microbiol. 2017; 122(4): 987–996. PubMed Abstract \| Publisher Full Text\n\nZhang FS, Lv YL, Zhao Y, et al.: Promoting role of an endophyte on the growth and contents of kinsenosides and flavonoids of Anoectochilus formosanus Hayata, a rare and threatened medicinal Orchidaceae plant. J Zhejiang Univ Sci B. 2013; 14(9): 785–792. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30252", "date": "31 Jan 2018", "name": "Vishal Kumar Deshwal", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article addressed to the effect of L-tryptophan and root exudates as inducer for the production of auxin, phytohormone by inoculated bacteria. Many works have been completed with PGP rhizospheric bacteria but naturally occurring indigenous bacteria can provide a better results in comparison to rhizospheric bacteria. In present article:\nIntroduction\nThis section of article is written in constructive manner to fulfil and provide the basic idea behind the use of bacteria for plant growth and phytohormone. It also summarize the role of endophytic bacteria for betterment of plants through various mechanism those are helpful for agriculture.\n\nMethods\nAuthor’s used basic (biochemical analysis) as well as molecular methods for the characterization of isolates that confirm the authenticity of identification of bacterial spp. Author mentioned the proper site of isolation with given altitudes so in future said work can be replicated with samples. All methods are explained properly with size of samples and other chemicals that satisfy the requirements if any one likes to duplicate the work in future.\n\nResult Characterization and phytohormone production results displayed by author are acceptable. Authors tried to evaluate the effect of endogenous bacteria, isolated from rice, on maize as well as effect of maize root exudates on bacteria and its colonization. Present article is a preliminary stage for the future prospects of isolated bacterial isolates as biofertilizer. Authors have to go further to test both isolates in field condition to use these endogenous isolates as biofertilizer.\nSuggestion: Author may add statistical analysis in Figure 2, 3 and 4 so results will show the significance of analysis.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "30813", "date": "27 Feb 2018", "name": "Loganathan Karthik", "expertise": [ "Reviewer Expertise Microbiology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic and work bought into notice are very interesting. The plant growth regulators produced by microorganisms, which could play a critical role in plant growth promotion for the promotion of sustainable agricultural practices.\nThe Abstract is well written and appropriate as per work done.\n\nThe Introduction section is well written.\n\nAll Results seems appropriate and is supported by straightforward tables and figures.\n\nThe Discussion is well presented and the findings were evaluated with current articles.\n\nMinor corrections:\nIn Abstract, Conclusions must be change in to Conclusion In Introduction part, the following sentence must be reframed - 80% of rhizospheric microorganisms naturally yield auxins as secondary metabolites due to the rich supplies of root exudates In results section: - Fig 1- the scale bar is missing - Fig 2,3,4- Remove the grid lines - Authors must carry out statistics for all results Results and discussion part must be combined Conclusion part is missing\nThe article can be indexed in F1000 journal with minor corrections as suggested.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-112
https://f1000research.com/articles/7-110/v1	25 Jan 18	{ "type": "Research Note", "title": "A structured process to create datasets with nutritional information", "authors": [ "Francisco Rodriguez-Montúfar", "Bayron Ordoñez-Buitron", "Diego Duran", "Angela Chantre-Astaiza", "Gustavo Ramirez-Gonzalez" ], "abstract": "There is a lack of datasets in Colombia that characterize the nutritional components and other similar information about food items. This study describes a structured process to develop datasets that captures the preferences and purchases of food items by a selected group of people. The datasets would classify products according to their sodium and sugar content. The outcome of this structured process would include three datasets, each with a different focus: the first contains data on food preferences, the second contains the purchase history according to the invoices obtained, and the third contains characteristics of the food items such as its brand, category, sodium and sugar content levels, among others.", "keywords": [ "dataset", "sodium", "sugar", "purchase invoices", "survey" ], "content": "Introduction\n\nIn this day and age, there is an impressive amount of data traffic that is generated and shared over the internet. Researchers can utilize thousands of photos, hours of video footage, and consumer data to create datasets1. Some datasets are used in research with a specific goal in mind, whereas other datasets are used to create data and store information for future investigations. Some datasets are freely published, while others are for restricted use.\n\nThere are several studies that use data to analyse taste preferences around online shopping2, music3,4, movies5,6 or social relations, for example 7. However, a study about people’s preferences for food items in supermarkets in Colombia faces challenges due to the lack of datasets freely available on this topic. Additionally, various products that are present in some public datasets8 are not available in Colombia.\n\nTo address these gaps, this study describes the process of creating and describing a dataset that contains information on the food preferences and purchases of a group of people living in Colombia. An important aspect of the dataset is describing the sodium9 and sugar10 content of each food product and featuring and sorting out the nutritional information available in the Colombian market.\n\n\nMethods\n\nAccording to the STROBE guidelines, we have taken the following into consideration.\n\nThe purpose of the study is based on capturing the preferences of users in self-service stores. The study was carried out in the cities of Popayán and San Juan de Pasto, Colombia, across two months, where part of this period was used for participant recruitment.\n\nA group of students, professionals and independent workers, all ≥ 18 years of age, accepted the invitation to participate in this research voluntarily, providing a signed agreement where they accepted to sharing their information as long as their identities would be protected and remained anonymous.\n\nAll data were analyzed and stored in text files available in the \"Variables and Data sources\" section. In that section, the structure and components are explained in more detail.\n\nThe study is exploratory and the aim is to obtain a dataset for future work. The general structure followed the principles outlined by Robert K. Yin11. Table 1 presents a summary of these elements.\n\nFigure 1 illustrates the process of data acquisition, carried out using two methods. The first method involved collecting preferences using a survey, and the second method involved the acquisition of purchase records with invoices. All purchases were made in self-service stores, focused particularly on food self-service stores. Data collection was implemented over a one-month period when participants were actively involved in the data collection process.\n\nData collection as described above was carried out through a survey, where people chose products based on their preferences. For this task, the Google Forms web tool was used, in which a series of questions were designed and classified into twelve sections. Participants were informed of the academic purpose of the survey, and the basic demographic data of each participant was registered. They identified their preferences out of the 708 food items presented in the survey. All items were classified into ten categories, created from observation of local self-service stores.\n\nTable 2 shows the 10 categories the items were classified under. Classification of the items aimed to have participants interact in a more comfortable and conscious way with the questions, attempting to keep the process from becoming tedious.\n\nThe survey was available for one month, and was available online. During the collection process, 215 people participated and shared their preferences and other demographic data.\n\nThe purchase history refers to a list of products purchased by a person within a period of time in a self-service store. 65 participants provided all of their purchase receipts for four weeks, in particular for food products. At the end of this period, all the invoices of the 65 people who participated in the study were collected. R-Studio v1.0.143.\n\n12 was then used to transcribe the products of interest, taking into account the number of submitted receipts, non-food products, and the number of times each user purchased each item.\n\nThe second part of Figure 1 illustrates how the information collected from the surveys was processed to construct the datasets. The process involved manually removing irrelevant information such as repeated surveys, inconsistent data, and non-focused responses in the user preferences section. For the participants’ purchase receipts, some information was also manually removed, since some receipts contained purchases other than food products. The previously filtered information in both datasets was anonymized by assigning numerical codes to the users and the products to protect users’ identities and classify all the products. All food items were classified based on their sodium and sugar content (based on WHO and FDA recommendations)9,10. Figure 2 shows the final data structure after organizing the information13.\n\nSurvey_items represents the preferences of the user and Purchase_items represents the purchases themselves, along with the characteristics of each product.\n\nThere are two columns in Table 3. The first column (“User Code”) shows the code assigned to each user, and the second column registers the products selected as each user’s favorite. Each user has one or more products registered in the table, where the first four numbers represent the type of product, and the last three numbers refer to the specific brand for each product.\n\nSimilar to the previous table, Table 4 presents the same two columns, and has an additional column, which shows the ranking, or the number of times a user has purchased that product divided by the number of shopping invoices for that user over the four week period.\n\nTable 5 has six columns, with each row representing a different product characteristic. Each product has an item code, the section to which the product belongs, the category to which the product belongs, brand, sugar content per 100 g, and sodium content per serving (classified into four levels, where 1 is the lowest and 4 is the highest).\n\nWritten informed consent was provided by all persons who volunteered in the research. Our study received approval in data management ethics according to the politics of the Telematics Engineering Group of the University of Cauca within the Electronics and Telecommunication Engineering Faculty. The proposed procedure is covered under approval number 8.4.2-90.14/274 of 2017. The study also complies with article 15 of the 1991 Colombian constitution on the right to privacy; and with the concepts of the Colombian Constitutional Court in Judgment No. T-414/92 of 1992 on the definition of data, computer freedom, and personal information.\n\n\nResults\n\nThe numbers in the second half of Figure 3 represent a scale for measuring sodium and sugar contents, based on the quantity of sodium and sugar that each product contained according to the nutritional table. To better understand the graph, we note again that there are four levels that represent the sodium content, and four levels that represent the sugar content, which generates 16 possible combinations that are color coded differently. For instance, the green circle with the number 11 indicates that the sodium and sugar contents are very low, whilst the red circle with the number 44 indicates a product with very high sodium and sugar content. The pie chart illustrates the percentage of products in each sodium and sugar classification. There is a higher percentage of products with high sodium and low sugar contents.\n\n\nConclusions\n\nThis work was carried out to construct a valid dataset with food items available in Colombia. Future academic studies can perform statistical analysis using the data collected. Using the information from the nutritional labels of food items, we classified products using aspects like sodium and sugar content, following WHO and FDA recommendations to inform us whether the products contain levels above or below the recommended levels.\n\nDataset 1: User preferences. This file contains two columns (User_Code, Item_Code), the first column User_Code is the code assigned to each user and the second column Item_Code contains the encoded product that the user prefers. DOI, 10.5256/f1000research.12979.d18837314.\n\nDataset 2: User purchasing. This file contains three columns (User_Code, Item_Code, Rating), the first column User_Code is the code assigned to each user and the second column Item_Code contains the encoded product that the user prefers and Rating is the value obtained from dividing the number of total product invoices by the number of times the user purchased a product. DOI, 10.5256/f1000research.12979.d18837415.\n\nDataset 3: Product characteristics. This file contains six columns (Item_Code, Category, Section, Code_Brand, Sugar_Level, Sodium_Level). Item_Code is the code assigned to each item and the other columns represent how they have been classified and coded according to their characteristics. DOI, 10.5256/f1000research.12979.d18837516.\n\nDataset 4: Products. This file contains three columns (No, Item_Code, Product\"), where Item_Code represents the code assigned to each product and Product represents the product type without specifying the brand. DOI, 10.5256/f1000research.12979.d18837617.\n\nDataset 5: Brands. This file contains three columns (No, Code_Band, Brand), Code_Brand represents the code assigned to each brand and Brand represents the brand of each product. DOI, 10.5256/f1000research.12979.d18837718.\n\nDataset 6: Categories. This file contains three columns (No, Code_Category, Category), Code_Category represents the code assigned to each category and Category represents the assigned to the product. DOI, 10.5256/f1000research.12979.d18837819.\n\nDataset 7: Sections. This file contains three columns (No, Code_Section, Section). Code_Section represents the code assigned to each section and Section represents the section in which a product can be found. DOI, 10.5256/f1000research.12979.d18837920.\n\nDataset 8: Sugar. This file contains three columns (No, Sugar_Level, Code_Sugar_Level). Sugar_Level classifies the products by sugar content and Code_Sugar_Level represents the code assigned to each level. DOI, 10.5256/f1000research.12979.d18838021.\n\nDataset 9: Sodium. This file contains three columns (No, Sodium_Level, Code_Sodium_Level). Sodium_Level classifies the products by sodium content and Code_Sodium_Level represents the code assigned to each level. DOI, 10.5256/f1000research.12979.d18838122.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nIbm.com: What is a data set? Reference Source\n\nMartinez-Pabon F, Ospina-Quintero JC, Ramirez-Gonzalez G, et al.: Recommending ads from trustworthy relationships in pervasive environments. Mobile Information Systems. 2016; 2016: 18. Publisher Full Text\n\nBertin-Mahieux T, Ellis DP, Whitman B, et al.: The million song dataset. In Proceedings of the 12th International Conference on Music Information Retrieval (ISMIR 2011). 2011. Publisher Full Text\n\nDefferrard M, Benzi K, Vandergheynst P, et al.: FMA: A Dataset For Music Analysis. ArXiv e-prints, 2016. Reference Source\n\nMaxwell Harper F, Konstan JP: The movielens datasets: History and context. Acm transactions on interactive intelligent systems (tiis). 2015; 5(4): 19. Publisher Full Text\n\nLeka O: IMDB movies dataset. 2016. Reference Source\n\nMartinez-Pabon F, Caicedo-Guerrero J, Ibarra-Samboni JJ, et al.: Smart TV-Smartphone Multiscreen Interactive Middleware for Public Displays. ScientificWorldJournal. 2015; 2015: 14, 534949. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPennacchioli D, Coscia M, Rinzivillo S, et al.: Explaining the product range effect in purchase data. 2013. Publisher Full Text\n\nFda.gov: Sodium in Your Diet: Use the Nutrition Facts Label and Reduce Your Intake. Acceded 06-10-2017. Reference Source\n\nWorld Health Organization: Sugars intake for adults and children. 2015; 2015: 59. Reference Source\n\nYin RK: Case Study Research. 2002.\n\nRStudio Team: RStudio: Integrated Development Environment for R. RStudio, Inc., Boston, MA, 2015.\n\nTechTarget Inc, Rouse M: Class diagram. 2017. Reference Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 1 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 2 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 3 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 4 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 5 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 6 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 7 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 8 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source\n\nRodriguez-Montufar F, Ordoñez-Buitron B, Duran D, et al.: Dataset 9 in: A structured process to create datasets with nutritional information. F1000Research. 2017. Data Source" }	[ { "id": "31841", "date": "03 Apr 2018", "name": "Georgina Gómez", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEven though the information obtained in this study could be used to further research, we consider that the simple attempt to provide a data base is not enough for publication. According to methodology, they should explain in detail about the sample, is socio-economic status a possible bias? How they were selected?\nThe paper lacks a discussion section to compare their results with other Colombian food data bases, or whether or not this can be extrapolated to other regions of the country.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "31839", "date": "17 Apr 2018", "name": "Irina Kovalskys", "expertise": [ "Reviewer Expertise NCD", "obesity", "nutrition", "population studies" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors had a good idea and implemented accordingly. Despite of that, scientific information is weak and reproducibility in not ensured with the information provided\n\n1. The goal of the study is not enough clear: Two phrases are used to describe the purpose:\na. “This study describes the process of creating and describing a dataset that contains information on the food preferences and purchases of a group of people living in Colombia. An important aspect of the dataset is describing the sodium and sugar content of each food product and featuring and sorting out the nutritional information available in the Colombian market.\nb. The purpose of the study is based on capturing the preferences of users in self-service stores.\n\nWhich is the exact main goal? Describing the process of the data set or communicating the results obtained?\n\n2. Methods a. This publication should be improved by giving much more detail about sample. Sample calculation and or sample size adequate to conclusions is missing. Purchasing habits is directly related to socioeconomic status, culture and habits. It is not possible to know the population represented in this sample. (characteristics of the population are not detailed)\nb. The authors say “All food items were classified based on their sodium and sugar content (based on WHO and FDA recommendations)”. Food groups and food classification should be described in detail to better understand Figure 3\n\n3. Conclusions I suggest to first clarify the aim of the study to obtain the main conclusions. It would be expected to summarize the main outcomes in this section\nI recommend the authors to write two scientific communications. One considering the process only and another one focusing on results\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-110
https://f1000research.com/articles/6-868/v1	12 Jun 17	{ "type": "Opinion Article", "title": "Clues to finding correlates of risk/protection for HIV-1 vaccines", "authors": [ "Marian P. Laderoute" ], "abstract": "Almost a decade later, we still do not understand why in the STEP trial (2008), males with pre-existing antibodies to the Ad5 vector were associated with initial increased risk of HIV-1 acquisition. Similarly, we have little conclusive evidence of why in the RV144 trial (2009), vaccination with the ALVAC-HIV/AIDSVAX B/E was associated initially with almost a 60% vaccine efficacy at year one, which waned over 42 months to 31.2%, and where females were more protected than males. Based on the literature and trial outcomes, it was deduced that the elusive correlate of risk/protection may pertain to a novel, potent, innate protector mechanism launched by alternatively activated macrophages, which is probably induced by viruses and female steroid hormones. It was also suggested this mechanism was not likely amenable to discovery using standard or traditional approaches.\n\nA plausible, candidate mechanism was identified with these characteristics, namely the production of human endogenous retrovirus–K102 (HERV-K102) particles, which occurs in, and generates, foamy macrophages in vitro. Accumulating clinical, biological and phylogenetic evidence supports its role in the antagonism of HIV-1 replication and/or in the prevention of HIV-1 acquisition. Thus, it will be important to examine HERV-K102 particle production, increased integration and envelop antibody production as candidate correlates of protection in HIV-1 vaccine trials, as well as in HIV-1 highly exposed seronegative cohorts and elite controllers. The results of such efforts may have important ramifications for the HIV-1 cure in addition to vaccines.", "keywords": [ "HIV-1", "novel", "correlates of protection", "HIV-1 vaccines", "HERV-K HML-2", "HERV-K102", "foamy macrophages", "innate immunity" ], "content": "Introduction\n\nIt has been recently stated that immune correlates of risk/protection for HIV-1 vaccines must be complex and/or reliant on the right combination of multiple types of immune responses, as correlates of protection have eluded investigators (Tomaras & Plotkin, 2017). However, the relatively low rates of acquisition of HIV-1 per exposure at less than 1 in 1000 for heterosexual transmission (Becerra et al., 2016), may instead argue that defense against HIV-1 in humans possibly involves a simpler and more potent mechanism than what has yet been elucidated or appreciated. Indeed, that a new scientific paradigm may be needed to advance the development of the HIV-1 vaccine has been recently proclaimed (Esparza, 2015). What then can we currently deduce about the characteristics of this unknown correlate of risk/protection, such as from the two informative HIV-1 preventative vaccine trials: one associated with increased HIV-1 transmission, known as the STEP trial (Buchbinder et al., 2008), while the RV144 trial that was associated with decreased HIV-1 transmission (Rerks-Ngarm et al., 2009)?\n\n\nThe elusive correlate is likely launched by activated macrophages\n\nIn any immune response, innate and/or adaptive, activated macrophages control the response. Accordingly, it follows that any risk or protection associated with HIV-1 vaccines must then relate to a key and so far, ill-defined macrophage activation pathway. Moreover, in HIV-1 acquisition, the transmitting/founder strains are generally CCR5-tropic and target macrophages (reviewed in Borggren & Jansson, 2015). Together these findings point to the likelihood that there exists a novel macrophage-based defense mechanism that itself determines whether HIV-1 will be acquired.\n\nThe Ad5 vector used in the STEP trial, highly targets liver Kupffer cells, which represent about 80–90% of the macrophages in the body (Khare et al., 2011). In the STEP trial, male uncircumcised participants with Ad5 antibodies were significantly at increased risk of transmission compared with those without vector antibodies (Buchbinder et al., 2008). This suggests that at the interface of the Ad5 vector with macrophages, the presence of bound Ad5 antibodies enriched in the local milieu may have somehow blocked the induction of the putative, gatekeeper/defense mechanism of macrophages. A possible candidate mechanism for this interference by antibody is tuftsin, a short peptide consisting of the sequence TKPR, which is released from bound IgG and is known to inhibit macrophage activation at higher concentrations, while at lower levels, it augments macrophage activation (Siemion & Kluczyk, 1999). Thus, it appears to be plausible that higher risk participants (uncircumcised men) with pre-existing Ad5 antibodies injected with an Ad5 vector could have experienced inhibition of the putative, novel macrophage defense mechanism such as by the local generation of tuftsin, thereby explaining their increased risk.\n\n\nOther salient clues to the characteristics of the novel host protection mechanism revealed from the informative HIV-1 vaccine trials\n\nAnother important clue relates to gender. In the STEP trial, women were notably at decreased risk of HIV-1 acquisition when compared with males (Buchbinder et al., 2008), whereas in the RV144 trial protection against HIV-1 acquisition was more evident in females than males (about 1.5-fold better) (Rerks-Ngarm et al., 2009). Together these findings suggest the novel, macrophage based protection mechanism is likely induced by female hormones.\n\nThat the risk/protection was only temporary in either trial, generally lasting 6 to 12 months after the last immunization (Buchbinder et al., 2008; Rerks-Ngarm et al., 2009), was consistent with an innate rather than adaptive immunity mechanism. In addition, following HIV-1 acquisition, the levels of CD8 and CD4 positive, CD38 positive, DR positive activated T cells are well recognized progression markers (Deeks et al., 2004), indicating adaptive immunity is unlikely to provide significant protection against HIV-1 replication. Accordingly, the key protection mechanism must be innate, but this does not necessarily rule out important contributions of intrinsic factors, such as APOBEC, which may antagonize HIV-1 replication and may also be upregulated with alternative activation of macrophages (Colomer-Lluch et al., 2016; Hartmann, 2017).\n\nSince both risk (Buchbinder et al., 2008) and protection (Rerks-Ngarm et al., 2009) were found with viral vectored vaccines, and not in the VAX003 and VAX004 HIV-1 vaccine trials, which instead involved proteins and adjuvants (reviewed in Shin, 2016), this raises the likelihood that viruses in general may activate the undefined macrophage-based defense mechanism, in addition to HIV-1.\n\nOverall, these observations point to the existence of a novel, potent innate HIV-1 protection mechanism induced by viruses and female hormones, which is launched by activated macrophages, possibly using alternative pathways, and that this activation may be sensitive to inhibition by locally bound antibodies, for example by tuftsin. Moreover, despite a considerable effort both inside and outside of the trials, convincing immune correlates of risk or prevention against HIV-1 have yet to be revealed. This failure raises the following notions about the mechanism. It is likely not addressed by traditional in vitro culture methods, not discoverable using conventional detection methods on participant samples, including microarray and genome wide association studies, nor is it likely present in the rhesus macaque. The critical question becomes, has such a novel, innate, potent, defense mechanism unique to human activated macrophages and difficult to study under standard conditions, been previously described?\n\nSurprisingly, the answer to this may be, yes.\n\n\nA novel, human-specific, foamy macrophage protection mechanism serendipitously discovered and relevant to protection against HIV-1 acquisition\n\nA novel, innate, viral defense mechanism unique to humans, associated with the production of foamy macrophages in vitro (Figure 1) was serendipitously discovered by scientists working at the Public Health Agency of Canada about 10 years ago, and fulfills all the above criteria for a defense mechanism launched by alternatively activated macrophages (Laderoute et al., 2007; Laderoute et al., 2015; Laderoute, 2015).\n\nLeft panel, H and E stain of day 11 cultured CB prepared by cytospin. Right panels, electron micrographs show vacuoles in the foamy macrophages contain large numbers of immature particles with envelope spikes. Blue arrow points to the cytoplasmic capsid assembly outside of the vacuole, typical of foamy retroviruses (Hütter et al., 2013). Left panel reproduced under a CC BY- NC 4.0 license (Laderoute et al., 2015). Right panels reproduced with permission from the AIDS journal (Laderoute et al., 2007).\n\nImportantly, this also includes initial evidence that it was associated with protection against HIV-1 acquisition in a female, HIV-1 highly exposed seronegative cohort (HESN), at the level of about 80% of the tested cohort (Laderoute et al., 2015). Moreover, protection in the infamous Nairobi HESN cohort (Fowke et al., 1996) is known to be temporary, as resistance to HIV-1 acquisition dwindled as early as 6 months to a year following abstinence from sex trade work (Kaul et al., 2001). Thus, this novel foamy macrophage mechanism, which might be implicated in the Nairobi HESN cohort, seems to meet a key criterion of temporary activity and waning after 6 months.\n\n\nA novel human, endogenous foamy-like virus generates foamy macrophages in vitro and may be remarkably potent\n\nThis putative defense mechanism launched by activated foamy macrophages in vitro (Figure 1) was characterized as human endogenous retrovirus K102 (HERV-K102, GenBank accession # AF164610) (Laderoute et al., 2015). HERV-K102 is an endogenous retrovirus unique to humans (Subramanian et al., 2011), and is the only HERV-K HML-2 group member which has been shown to be replication competent in vivo and as associated with viremia (Laderoute et al., 2007). As extensively reviewed elsewhere (Laderoute et al., 2015), it has hallmark features of non-pathogenic, foamy retroviruses. Like foamy viruses, its genomes are predominantly DNA rather than RNA (Laderoute et al., 2007) and replication involves budding into vacuoles rather than the cell surface, which renders cells foamy (Figure 1). It is important to note that the particles are released by cell lysis of the foamy macrophages and that foamy macrophages do not express HERV-K102 envelope at the cell surface (unpublished studies; Marian Laderoute). When cord blood mononuclear cells (CB) were cultured in IMDM rather than RPMI media, HERV-K102 spontaneously replicated, generating high levels of foamy macrophages (Laderoute et al., 2007; Laderoute et al., 2015). Others have similarly reported the induction of foamy macrophages when CB was cultured in IMDM (Stec et al., 2007). HERV-K102 appears to be the only HERV-K HML-2 element so far shown to be naturally replication competent in vitro (Laderoute et al., 2015) and in vivo (Laderoute et al., 2007).\n\nHERV-K102 particles released by freeze-thaw cycles of cultured CB cells, induced rapid and complete cell lysis of MRC-5 cells at 24 hours, which was not demonstrated for other cell lines tested (unpublished study; Marian Laderoute). This was expected as foamy viruses are well known to produce rapid cell lysis of some, but interestingly, not all fibroblastic cell lines (Linial, 2001). However, it remains to be determined which cell death pathway (Duprez et al., 2009) might mediate this remarkably rapid cell lysis, if integration is needed, and more critically, if HERV-K102 particles might similarly lyse HIV-1 infected cells. It is notable that HERV-K102 also appeared to be strongly induced in vivo increasing from no particles detected to 2.55 × 1011 cDNA containing particles per ml of plasma within 84 hours (unpublished study; Marian Laderoute). High levels of particles at 1010 to 1012 per ml of plasma were frequently found in patients viremic for various bloodborne pathogens, but maximum levels in HIV-1 patients were notably 7 to 8 log-fold downmodulated in comparison (Laderoute et al., 2007). It can be estimated from previously reported data that in HIV-1 patients there may only be on average about 8,200 DNA containing particles per ml of plasma corresponding to HERV-K102 (Laderoute et al., 2007), which has been substantiated by others but as demonstrated by the detection of excess HERV-K HML-2 transmembrane envelope DNA but not RNA sequences on isolated particles as compared with normal healthy controls. (Bhardwaj et al., 2014). It should also be noted that two groups have reported that HERV-K HML-2 particles with RNA genomes were not demonstrable in HIV-1 patients (Bhardwaj et al., 2014; Karamitros et al., 2016), consistent with the notion that HERV-K102 transcripts in particles are predominantly or exclusively DNA (Laderoute et al., 2007). Taken together these results may suggest only HERV-K102 particles with DNA genomes are significantly produced in HIV-1 patients, but this needs to be more carefully addressed. Thus, overall, based on rapid induction, the high levels of particles that can be produced both in vitro (Figure 1) and in vivo (Laderoute et al., 2007), and potentially swift cell kill by particles (unpublished study; Marian Laderoute), HERV-K102 particles could comprise a potent innate immune mechanism launched by foamy macrophages. However, upon HIV-1 acquisition, HERV-K102 particle production and/or release appear to be strongly inhibited.\n\n\nHERV-K102/HML-2 expression associated with HIV-1 infection or exposures to HIV-1\n\nIn vitro, and despite suboptimal conditions by culture of the cells in RPMI, several research groups have confirmed that HERV-K102 is induced by HIV-1 (Brinzevich et al., 2014; Vincendeau et al., 2015). Moreover, HERV-K102 may be the only human specific, full length HML-2 element induced by HIV-1 and/or Tat (Gonzalez-Hernandez et al., 2012). While the envelope of HERV-K18 and a consensus sequence for HERV-K HML-2 were able to pseudotype HIV-1 virions, interestingly HERV-K102 did not (Brinzevich et al., 2014; Lee & Bieniasz, 2007). That HIV-1 may be pseudotyped by HML-2 envelope raises the notion that such pseudotyped particles could help explain, in part, the altered tropism for macrophages bearing the CCR5 coreceptor, which is commonly used by transmitting/founder strains (Borggren & Jansson, 2015). If this is indeed the case, it would also help explain why vaccination against HIV-1 envelope generally fails to prevent HIV-1 acquisition (reviewed in Shin, 2016), or why passive immunization with HIV-1 envelope specific, broadly neutralizing envelope antibodies failed to significantly control viremia upon antiretroviral treatment interruption (Bar et al., 2016; Caskey et al., 2017).\n\nThe mean HERV-K102 pol copy number in the genomes of the HESN, as demonstrated on DNA extracted from plasma, was elevated about 5-fold above the genomic levels of normal healthy adults (p<0.0005) (Laderoute et al., 2015). This is consistent with high integration levels reported for foamy viruses in hematopoietic cell lines (Meiering et al., 2000). This implied very high HERV-K102 particle production likely occurred in the HESN cases. In direct contrast, there was no evidence for increased mean genomic copy number above normal healthy controls for North American individuals already infected with HIV-1, irrespective of their use of anti-retroviral therapy (Laderoute et al., 2015). However, evidence of the activation of this macrophage-based defense system was demonstrated in about 96% of HIV-1 patients which includes particles and/or HERV-K102 surface unit envelope specific antibodies (post hoc analysis, Laderoute et al., 2007). Thus, as might be expected, individuals protected against HIV-1 acquisition may produce high numbers of HERV-K102 particles reflected by increased integration, but upon its acquisition, HERV-K102 particle production was strongly downmodulated with no evidence of increased integration of HERV-K102 sequences (Laderoute et al., 2007; Laderoute et al., 2015).\n\nIn potential substantiation of an important role of HERV-K102 in the control of HIV-1 replication, HERV-K HML-2 gag and envelope RNA expression in peripheral blood mononuclear cells (PBMCs) in HIV-1 patients were shown to be inversely correlated with T cell activation markers (Ormsby et al., 2012). Since it is known that activated T cells correlate with HIV-1 progression (Deeks et al., 2004), this implies HML-2 expression generally, and by proxy HERV-K102 activation, may antagonize HIV-1 replication. This argument is further strengthened by recent evidence that suggests the newer HERV-K HML-2 elements containing LTR5Hs (which include HERV-K102) are upregulated in CD11c+ myeloid dendritic cells isolated from HIV-1 patients, whereas, in normal healthy controls, the older LTR5A and LTR5B bearing HML-2 elements prevailed (Young et al., 2014).\n\nBoth antibodies and T cell responses to HERV-K HML-2 and/or HERV-K102 envelope have been demonstrated in HIV-1 and breast cancer patients (reviewed in Laderoute et al., 2015). A T cell clone isolated from an elite controller, which recognized a peptide identical to HERV-K102 envelope, was shown in vitro to specifically eliminate cells infected with various HIV and SIV strains (Jones et al., 2012). Remarkably, a monoclonal antibody made to HERV-K102 envelope could directly provoke apoptosis in vitro and in vivo (Wang-Johanning et al., 2012). This might suggest that the expression of HML-2 envelope on the surface of virally infected or transformed cells, but which is not found on normal cells, plays a more active role in innate host protection than merely as a surrogate marker. These findings may also further document the unexpected potency of this innate protector mechanism against HIV-1, which unlike adaptive immunity, remarkably functions irrespective of the hypervariability of HIV-1, quasi-species and/or strains of HIV-1 or lentivirus involved.\n\n\nGender differences in HERV-K HML-2 activation\n\nIn terms of other characteristics of the defense mechanism deduced earlier from the informative HIV-1 vaccine clinical trials, combination female steroid hormones (estrogen then progesterone) have been shown to stimulate the expression of HERV-K HML-2 (Ono et al., 1987). In a recent meta-analysis, while the use of various progestins for oral contraception were associated with a significantly increased adjusted hazard ratio of HIV-1 acquisition over women who did not use contraceptives, the combined oral contraceptive was not (Morrison et al., 2015). This is consistent with the notion that women of child-bearing age (and not on progestins), may be more protected against HIV-1 acquisition compared with male counterparts possibly through regular, monthly induction of the HERV-K102 protector system by combined estrogen and progesterone.\n\n\nHERV-K102 particle production is not addressed by standard methodology\n\nThe identification and elucidation of correlates of protection against HIV-1 have been challenging. Overall the failure to identify HERV-K102 particles pertains largely to the notion that its presence is, more often than not, overlooked or not addressed by standard methodological approaches. For example, because HERV-K102 is unique to humans (Subramanian et al., 2011), it is absent from animal models, such as macaques and rodents, which are commonly used for vaccine or immunological investigations. In addition to HERV-K102 and HML-2 being inhibited when PBMCs or CBs are cultured in the more traditional RPMI media invariably used by immunologists (Argaw-Denboba et al., 2017; Laderoute et al., 2015), HERV-K102 activation is also blocked by the depletion of CD14 + cells from PBMC, and also by the addition of PHA and IL-2 to cultures performed in IMDM (unpublished studies; Marian Laderoute). Accordingly, it may not be a co-incidence that the conditions that block HERV-K102 particle production in vitro are those that instead are commonly employed to demonstrate HIV-1 infectivity, such as purified T cells activated with PHA and IL-2 cultured in RPMI. Indeed, these observations would be consistent with the possibility that HERV-K102 particles may antagonize HIV-1 replication in vitro; however, importantly this needs to be directly examined.\n\nThe detection of the presence of HERV-K102 particles also eludes other common approaches utilized for investigations. For example, detection of particles in plasma requires an alternative isolation strategy seldom employed by retrovirologists. It requires DNA and not RNA isolation from plasma (Laderoute et al., 2007), where the use of DNAse would be contraindicated. As well, genome wide association studies and microarray analysis typically exclude highly repetitious sequences (Baranzini et al., 2010; Held et al., 2003, respectively) to which this element belongs. Accordingly, HERV-K102 particle production appears to have eluded the field due to the difficulty in demonstrating its presence using standard or traditional approaches.\n\n\nTuftsin may modulate HERV-K102 DNA production in vitro\n\nRelevant to the increased risk of HIV-1 acquisition related to Ad5 antibodies in the STEP trial (Buchbinder et al., 2008), at a high concentration (2 mg/ml), tuftsin inhibited the production of HERV-K102 DNA in cultured CB by 53%, while at a lower concentration (200 ng/ml), tuftsin enhanced the replication of HERV-K102 pol containing DNA over normal genomic levels by 237% (unpublished study; Marian Laderoute). Thus, it seems as a protector mechanism launched by alternatively activated macrophages, HERV-K102 particle production might be subject to modulation by tuftsin and thus possibly relevant to the adverse outcomes of the STEP trial. Clearly, further investigation of the mechanisms of how pre-existing antibodies were associated with adverse outcomes in the STEP trial appears to be warranted.\n\n\nHERV-K HML-2 activity has been phylogenetically associated with decreased integration of orthoretroviruses in hominins\n\nAccumulating phylogenetic evidence is consistent with a potential role of HERV-K HML-2 in limiting invasion by orthoretroviruses (Magiorkinis et al., 2015). Ancestral HML-2 elements emerged about 10.3 million years ago (Mya) (Subramanian et al., 2011). There has been a striking decline of insertions of ERVs in the last 10 My in the genomes of all sequenced hominids (great apes and gibbons), but not in old world monkeys (baboons and macaques), particularly regarding HERV-H (Magiorkinis et al., 2015). HERV-H makes up 88% of all the ERV integrations into the human genome in the last 30 My and became extinct over the past 10 My. HERV-H is a gammaretrovirus, which integrated around 45 to 60 Mya and has about 962 copies in the human genome (Chuong et al., 2016). HERV-K, with 10 groups in the clade, only one of which is HML-2, on the other hand, entered the genome of ancestral catarrhines about 32 to 44 Mya, after the split from New World monkeys and before the split of hominids from the Old World monkeys (Kim & Han, 2015). The sister lineages of HERV-K in most other catarrhines appear to have become extinct. Most remarkably, the HERV-K HML-2 group in humans is the only HERV-K that has continued to replicate since the origin of the catarrhines (Magiorkinis et al., 2015).\n\nAccordingly, since phylogenetic evidence supports an association of HERV-K HML-2 activity with protection against integration of orthoretroviruses, this may help substantiate the claim that modern day HERV-K102 particles, along with expression of proteins from other HML-2 elements, might antagonize HIV-1 replication and/or prevent its acquisition.\n\n\nThe origins of HERV-K102 in humans\n\nSomewhat ironically, humans apparently acquired the HERV-K102 defense mechanism possibly between 500,000 and up to 2 Mya (Romano et al., 2006; Subramanian et al., 2011), from the same source of the modern HIV-1 pandemic strain; namely, chimpanzees.\n\nThe Homo-Pan split has been estimated at 6.6 Mya (Magiorkinis et al., 2015) or earlier at 7-8 Mya (Langergraber et al., 2012). As mentioned, the HERV-K HML-2 elements originated in primates about 10.3 Mya and the CERV-K102 sequence (DQ112149), which is 97% identical to HERV-K102, was estimated to have integrated into chimpanzees at a non-orthologous position about 10 (+/- 3.3) Mya (Romano et al., 2006). Lentiviruses may have been active in primates since the divergence of chimpanzees and humans (Katzourakis et al., 2007; Sawyer et al., 2004). Moreover, it has been suggested the ancestor to HIV-1 may have arisen in chimpanzees about 4 Mya (Gifford, 2012). Since, it has been reported that subsets of chimpanzees with chronic HIV-1 infection showed progression analogous to humans, including greater expression of CD38 in CD8+ HLA-DR+ T cells (O’Neil et al., 2000), this raises the notion that an HERV-K102 ancestor, as a potential antidote for HIV-1 infection may have been selected through evolution in chimpanzees before it was acquired by humans. Accordingly, it is possible over about a 2 to 3.5 million-year window or longer, the HERV-K102 ancestor may have adapted to an HIV-1 like ancestor lentiviruses in chimpanzees prior to its acquisition by humans.\n\n\nConclusion\n\nThis inquiry has led to the notion that HERV-K102 particle production, which generates foamy macrophages, appears to fulfil the requirements of a deduced candidate correlate of protection against HIV-1 acquisition. Moreover, this candidacy has been strengthened by biological, clinical and phylogenetic evidence, including that which implies HERV-K102 particles may be associated with protection against HIV-1 acquisition. That conversely, acquisition of HIV-1 would be associated with significantly log-lower levels of HERV-K102 particles, would be anticipated and was observed. Given also the preliminary evidence that tuftsin could block the replication of HERV-K102 in vitro, suggests the blocking of the same mechanism, such as by Ad5 antibodies in the STEP trial shown in males at higher risk, could plausibly account for the increased risk observed in this informative trial. Finally, that the host source of this remarkable innate protection mechanism appears to be the same as that for pandemic strains of HIV-1 would strengthen its authenticity, especially given the likelihood of millions of years of co-evolution of HERV-K102 and HIV-1 in chimpanzees. Overall, the available evidence substantiates that a special antagonistic relationship exists between HIV-1 and a foamy-like virus, HERV-K102.\n\nAccordingly, it will be extremely important to prioritize the testing of human endogenous retrovirus K102 (HERV-K102) particle production, integration, and/or envelope specific antibody production to prove or disprove it as a correlate of risk/protection on actual STEP and RV144 clinical trial participants (Figure 2). Exploratory studies in other HESN cohorts and in elite controllers may also serve to further strengthen the correlation. No less significantly, the clinical ramifications of pseudotyping of HIV-1 virions by HML-2 envelope needs to be addressed as it may also help explain in part the failed vaccine and cure attempts.\n\nAdapted from Figure 1. Note that in the STEP trial, in addition to Ad5 antibodies, Th1 cytokines, such as might be released by Ad5 or HIV-1 antigen responsive T cells, could potentially also block HERV-K102 particle production in macrophages since PHA with IL-2 blocked HERV-K102 particle production in vitro (unpublished study; Marian Laderoute). Consistent with this notion, lower serum levels of IL-2, IFN-γ, and GM-CSF were recently demonstrated in a cohort of HESN over HIV-1 unexposed controls (Jaumdally et al., 2017), and IFN-γ levels were higher in HIV-1 positive over HIV-1 negative individuals (Yong et al., 2016).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed. While the author was named as one of the inventors in patent applications for the discovery of HERV-K102 as a replication competent foamy-like virus and for envelope specific antibodies either of which may have applications in the field of infectious diseases, by policy at the Public Health Agency of Canada, public servants have no rights nor entitlements.\n\n\nGrant information\n\nThe author declares that no grants were involved in supporting this work. However, unpublished studies cited were previously supported by funding from the Blood Safety Program at the Public Health Agency of Canada and in part by an Innovative Research grant from the Office of the Chief Scientist at Health Canada.\n\n\nReferences\n\nArgaw-Denboba A, Balestrieri E, Serafino A, et al.: HERV-K activation is strictly required to sustain CD133+ melanoma cells with stemness features. J Exp Clin Cancer Res. 2017; 36(1): 20. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "26997", "date": "27 Nov 2017", "name": "Patricia E Fast", "expertise": [ "Reviewer Expertise Vaccines", "HIV epidemiology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDr. Laderoute has advanced a novel and interesting hypothesis, that an innate immune state associated with the virus HERV-K 102 can, in some circumstances, prevent or reduce the risk of HIV infection by a mechanism of infection of macrophages and activation of innate immunity. Dr Laderoute also proposes that this mechanism can go wrong in such a way as to increase risk and finally that vaccination with HIV vaccine candidates can influence this interaction or vice versa. A number of facts or interpretations are marshalled to support this hypothesis.\n\nThe main weakness of the paper is that it discusses a proposed causal relationship but, at many points in the argument, mentions only one interpretation of existing data and ignores alternative explanations, even when there are reasonably strong data supporting those explanations (for example, the elegant demonstration of correlate of risks in RV144 described by Haynes, Kim, Tomaras, Zolla-Pazner and others).\n\nThe basic postulate proposed is that an endogenous retrovirus, HERV-K102 might interact with macrophages, particularly Kupffer cells, and that the innate immunity induced would temporarily (6 months) alter host susceptibility to HIV infection, in a way that could interact with HIV vaccine effects. Part of this argument is that ‘macrophages’ are involved in initiation or control of adaptive immune responses. However, the dendritic cells that are intimately involved in initiating immune responses, differ from Kupffer cells, and this difference is not discussed.\n\nHormonal influence on the HERV-K102 link to innate immune states are postulated because in efficacy trials of one HIV vaccine, there appeared to be a gender effect on the results (the difference in efficacy between women and men in RV144 was not statistically significant). In fact the exposure, risk factors and baseline incidence differed between men and women in the two trials of the Ad5-vectored vaccine, STEP and Phambili, and it cannot be concluded from those data alone that there is a hormonal influence on susceptibility, let alone that is linked to endogenous retroviruses.\n\nThe cohort of highly-exposed female sex workers in Nairobi is also cited as potentially protected by transient innate immune mechanisms related to infection of macrophages with the endogenous virus. This putative immunity fades within months after the women cease sex work—it seems unlikely that a mechanism based on co-infection with HERV-K102 would be influenced by ending sex work or that the endogenous retrovirus infection somehow depends upon continued sex work. If the HERV-K102 virus-related mechanism fades after 6 months, that should be 6 months from the time of infection with the HERV-K102 virus (which is unknown), not 6 months after ceasing sex work.\n\nNo evidence is cited to link the virus infection itself or its interaction with macrophages to vaccination with HIV vaccines (a suggestion is made that the peptide tuftsin could be involved, but the link of tuftsin to HIV vaccines is not clear to me). To Dr Laderoute’s credit, she points out that a direct examination of this prediction is needed to test the hypothesis.\n\nIn summary, the endogenous retrovirus is fascinating, and its potential interactions with HIV may be worthy of study. This is clearly an opinion paper, so some latitude in the discussion is permissible, but the paper does not make a sufficient case for its hypothesis. Highly selective interpretations of data in the literature have been made to link that virus with protection from HIV infection, and alternate explanations are not explored adequately. The postulated alteration of the effects of HIV vaccines on risk of infection requires several logical leaps, each of which has alternate explanations, and the range of evidence is not discussed in sufficient detail. The paper could be improved by limiting its scope and providing a much more comprehensive and balanced argument.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? No\n\nAre arguments sufficiently supported by evidence from the published literature? No\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? No", "responses": [ { "c_id": "3341", "date": "25 Jan 2018", "name": "Dr. Marian Laderoute", "role": "Author Response", "response": "First I would like to sincerely thank Dr. Patricia Fast for taking the time to read through this opinion-hypothesis paper as the concept of correlates of protection (‘true’ or not) against HIV-1 is vitally important for the derivation of HIV-1 prevention vaccines as well as cure. In hindsight, I wholeheartedly agree that I should have had more discussion on proposed correlates of protection from HIV-1 vaccine prevention trials and why these have been rejected by others and myself. This has been corrected in the revision and importantly includes a vigorous discussion on why adaptive immunity mechanisms are unlikely to prevent HIV-1 acquisition (i.e., the elephant in the room) substantiated directly by the finding that there is no evidence in humans to defend this approach. The concept that the HERV-K102 virus unique to humans represents a ‘virus anti-virus response’ (in this case a foamy retrovirus anti-lentivirus response) and is part of innate immunity which generates foamy macrophages, is hard to grasp because it is so revolutionary. So now I have made this point of it being a ‘virus anti-virus response’ crystal clear not only in the abstract, but in the introduction, and have also tried to present the concept of the HERV-K102 virus being anti-HIV-1 more clearly. I think this may be why publication in F1000 Research with its open review can be extremely valuable for the communication of completely novel, if not totally foreign ideas. As a writer, I have to know why readers are not ‘getting it’ so that I can try to explain the concepts better. A main weakness is the poor discussion of correlates of protection from HIV-1 vaccine prevention trials and only mentions one interpretation of existing data. The revision now incorporates a lengthy discussion of correlates of protection from HIV-1 vaccine prevention trials and why one must conclude that a surrogate correlate of protection or a ‘true’ correlate of protection protecting against HIV-1 acquisition has not been demonstrated in the HIV-1 vaccine prevention trials. Indeed, a true correlate of protection has not been convincingly demonstrated not only in HIV-1 vaccine prevention trials (over 200 attempts) but also in studies of HIV-1 exposed seronegative cohorts (Safrit & Koff, 2016). Macrophages such as Kupffer cells are distinct from dendritic cells (DCs) and Dr. Fast implies only DCs are involved in initiating immune responses. This difference was not discussed. According to a recent review article, developmental studies in mice suggest DCs originate independently from monocytes and tissue macrophages, and for a long time these subsets of antigen-presenting cells were thought to overlap (Collin M et al., Immunology 2013). However, both cell types are antigen-presenting cells and are still studied and are relevant in terms of activating innate and adaptive immunity as derived and studied from human blood (Sander J et al., Immunity 2017; Metcalf TU et al., J. Immunology 2017). Kupffer cells constitute 80-90% of the tissue macrophages in the body and are essential to innate and adaptive immunity (Li P et al., Molecular Immunology 2017). The point being made in the paper is that the Ad5 vector targets Kupffer cells (which are macrophages) and the foamy macrophages which appear to produce HERV-K102 particles are most likely CD14 positive (as elimination of the CD14 positive cells from cord blood mononuclear cells abrogated HERV-K102 particle production, the production of foamy macrophages, the accumulation of HERV-K102 cDNA and the increase in HERV-K102 pol gene copy number in genomic DNA (unpublished data)). These foamy macrophages are morphologically distinct from all others and resemble those produced under similar conditions from cord blood mononuclear cells by Stec M et al., (J. Leukocyte Biology, 2007) which were characterized as CD14++ CD16+ macrophages which showed enhanced expression of CD11b, HLA-DR and CCR5. CCR5 is critical to the HIV-1 story, and thus, foamy macrophages may be very relevant to the proposed protective innate mechanism. Gartner et al., (Science, 1986) were first to elucidate that acquisition of HIV-1 first involves infection of macrophages and this has been followed by many publications regarding the R5 versus X4 strains (eg. see reviews Arif MS et al., 2017; Naif HM, Inf Dis Rep 2013), wherein CCR5-tropic (R5) viruses are preferentially transmitted by all routes of infection (Shaw GM & Hunter E, Cold Spring Harb Perspect Med 2012). The CD14++CD16+ monocytes express the highest levels of CCR5 than CD14++CD16- or CD14lowCD16+ and are preferentially infected by HIV (Ellery PJ et al., J Immunology 2007). In contrast it should be noted that DCs express CD4 but are poorly sensitive to HIV-1 infection (Chauveau L et al., J. Virol 2017). Moreover , SAMHD1 in DCs not only impacts HIV-1 replication but also antigen presentation (Ayinde D et al., J. Virol 2015). In a recent study involving humanized mice, it was the CD14+CD16+ monocytes/macrophages rather than CD14-CD16+ dendritic cells which increased during early infection and harbored integrated HIV-1 DNA such as found in spleen (Arainga M et al., Sci.Reports 2016). It is the monocyte/macrophage which serves as a viral reservoir during HIV-1 infection (Arainga M et al., Retrovirology 2017). Accordingly, the issue of dendritic cells versus macrophages was not discussed as DCs may not be so relevant to HIV-1 infection and pathogenesis. However, in fairness, it has been suggested in mucosal studies that the vaginal myeloid dendritic cells may transmit founder HIV-1 (Shen et al., 2014). Gender differences on VE in the HIV-1 prevention vaccine trials was only shown in one of two trials. There were insufficient HIV-1 infections in the STEP trial to calculate VE. The difference in VE between women and men in RV144 was not statistically significant. While the authors (Rerks-Ngarm et al., NEJM 2009) state that subgroup analyses revealed no significant heterogeneity in vaccine efficacy according to baseline variables (Table 2), p values were not provided for the subgroup analyses in Table 2. A discrepancy between the MITT VE 95% CI reported in Table 2 (n=15,948) and the abstract (n=16,395) was noted for “all subjects” whereby in the analyses of Table 2 there were 447 less participants (missing 237 in the vaccine arm of which 158 were male and 210 in the placebo arm of which 146 were male, from Table 1). Potential discrepancies in the lower and upper boundaries of the 95% CI reported for the two subgroup analyses by gender when compared with all subjects particularly the lower boundary (negative values for each of the sex subgroups but positive for the ‘all subjects’ category) might imply an error was made in the reported CIs. For example, it might be possible that the lower boundary for the CI of the female subgroup analysis might have been positive, suggesting statistical significance. Accordingly, a VE of 38.6% in the female subgroup when compared with 25.8 % in the male subgroup may or may not have been statistically significant and needs further clarification. Nevertheless, presently, one could refer to this finding as a trend towards potential significance. This fine point has been rectified in the revision. There is no evidence from the vaccine prevention trials that endogenous retroviruses are hormonally regulated or that susceptibility to HIV-1 infection is influenced by HERVs. This is correct because it was not tested. The point being made is to test for HERV-K102 particles and/or integration levels by gender subgroups (and by type and use of contraceptives) to characterize if HERV-K102 might be involved as a correlate of protection. Co-infection with HERV-K102, the time of infection which is unknown, cannot explain why in an HESN cohort involving sex work this putative innate immunity could fade such as by 6 months by ending sex work. There is no co-infection of the host by HERV-K102 as it is not an exogenous virus. The induction of HERV-K102 virus which is unique to humans, is a host ‘virus anti-virus’ response. The HERV-K102 virus located on chromosome 1 is found in all humans. It becomes active and produces particles in large numbers of foamy macrophages in response to viruses (Laderoute et al., AIDS 2007; Open AIDS J, 2015). Elite controllers have strong cellular and antibody responses to HERV-K HML-2 Gag (de Mulder M et al., Retrovirology 2017) not present in viremic non-controllers nor healthy controls. While particles can be made at extremely high levels in patients with infected with blood borne pathogens (commonly up to 1011 particles per ml of plasma), in patients with HIV-1 infection the mean particle production is around 8,300 particles per ml (maximum levels are 6-7 logs reduced) and is found in about 72 % of HIV-1 patients. In the Nairobi HESN cohort, evidence of past high levels of HERV-K102 particle production was provided by showing on DNA isolated from plasma, that the mean genomic copy number was significantly (p=0.0005) elevated by about 5 fold above controls (the latter shown not to have any HERV-K HML-2 particles in plasma including HERV-K102). Increased integration was not found in HIV-1 infected patients (which were not statistically different from uninfected healthy controls). There can be a very rapid induction of large numbers of HERV-K102 particles in vitro and in vivo, the latter reaching 2.55 x 10 11 particles per ml of plasma in 84 hours. Studies by others have shown that antibodies to HERV-K HML-2 antigens wane within 6 months with germ cell tumor remissions (Kleiman A et al., Int J Cancer 2004). In the absence of exposure to pathogens (or cancer), the stimulus for HERV-K102 particle production and thus antibody/T cell responses might wane, consistent with findings in normal healthy controls as reported by many groups. Thus, the finding by Kaul et al, 2001 that resistance to HIV-1 acquisition fades as early as 6 months following sex work interruption in the Nairobi HESN cohort could be related to decreased HERV-K102/HML-2 activation due to lack of stimulation by or exposure to intracellular microbes. No evidence is cited to link the virus infection itself (HERV-K102) or its interaction with macrophage to vaccination with HIV vaccines. There is no exogenous infection for an endogenous retrovirus. It is proposed that vaccination with viral vectors used in the 3 informative HIV prevention trials and not those lacking vectors for the priming may be analogous to exposures to viruses which might preferentially activate HERV-K102 particle production along with HML-2 activation over adaptive immunity. There seems to be reciprocal activation of adaptive immunity with the HERV-K102/HML-2 response. For example, in vitro, the addition of PHA and IL-2 has been shown to completely block HERV-K102 particle production under conditions permissive for HERV-K102 particle production (i.e., culture in IMDM and not RPMI media, more than a dozen replicates unpublished data). Similarly culture in RPMI blocks HERV-K102 particle production (unpublished observations) but allows adaptive immunity mechanisms (see also Argaw-Denboba et al., 2017 for HERV-K activation interference). As well, RNA for HERV-K MHL-2 gag and env was found to be inversely correlated with T cell activation markers when tested on PBMCs from HIV-1 patients, also implying an inverse correlation of HML-2 responses with adaptive immunity. Moreover as discussed in the revised manuscript, there were non-specific effects of the viral vectors unrelated to HIV-1 antigen specificities (STEP, Huang et al., 2014; STEP, Migueles et al., 2011, and see discussion in Tomaras & Plotkin, 2017)." } ] } ]	1	https://f1000research.com/articles/6-868
https://f1000research.com/articles/7-108/v1	25 Jan 18	{ "type": "Review", "title": "Associative memory cells and their working principle in the brain", "authors": [ "Jin-Hui Wang", "Shan Cui ", "Shan Cui " ], "abstract": "The acquisition, integration and storage of exogenous associated signals are termed as associative learning and memory. The consequences and processes of associative thinking and logical reasoning based on these stored exogenous signals can be memorized as endogenous signals, which are essential for decision making, intention, and planning. Associative memory cells recruited in these primary and secondary associative memories are presumably the foundation for the brain to fulfill cognition events and emotional reactions in life, though the plasticity of synaptic connectivity and neuronal activity has been believed to be involved in learning and memory. Current reports indicate that associative memory cells are recruited by their mutual synapse innervations among co-activated brain regions to fulfill the integration, storage and retrieval of associated signals. The activation of these associative memory cells initiates information recall in the mind, and the successful activation of their downstream neurons endorses memory presentations through behaviors and emotion reactions. In this review, we aim to draw a comprehensive diagram for associative memory cells, working principle and modulation, as well as propose their roles in cognition, emotion and behaviors.", "keywords": [ "Associative memory cell", "synapse", "neuron", "learning", "cognition", "brain" ], "content": "\n\nAssociative learning is an experience where multiple exogenous signals are jointly acquired through sensory systems. Associative memory involves the integration and storage of these associated signals in nerve cells, whose achievement can be proved by memory retrieval (recall and representation) via behaviors. Associative learning and memory is a common form of information storage for cognition throughout life1–4. In associative learning and memory, characteristic signals of each object or an environment are detected by distinct sensory modalities and cortices. These cross-modal signals are integrated for their associative storages. For instance, an orange is detected by the olfactory system for aromatic odor, the visual system for shape and color, the taste system for sweetness, the auditory system for name and so on. After associative memory forms, one of these signals induces the recall of other associated signals, and these signals are retrieved reciprocally, i.e., a signal induces the recall of its associated signals, or the other way around. How do sensory cortices integrate these signals, during initial associative learning, for associative storage, such that individuals can describe the object or environmental features? How are these associatively stored signals retrieved distinguishably in the brain? Although plasticity in the strength of synaptic connections and neuron activity is presumably a cellular mechanism for learning and memory5–8, the potentiation or depression of synaptic and neuronal activities in a pathway cannot account for the integrative storage of associated signals. Current reports show that nerve cells are reformed to be associative memory cells based on new synapse innervations from co-activated sensory cortices for the integrative storage of associated signals2,3,9–15.\n\nIn cognitions, logical reasoning, associative thinking and integrative imagination to these stored exogenous associated signals generate their secondary integrations, which can be stored as well as subsequently be recalled and represented. The storage of these secondary integrated signals (i.e., secondary associative memory) are essential for comparison, computation, decision making and planned intention under the consciousness condition3. Although the memory occurs presumably in the prefrontal cortex, hippocampus, amygdala and so on16–28, whether these studies are involved in the second grade of memory based on information storage in primary associative memory cells of sensory cortices remains unknown3. Current reports show that secondary associative memory cells are detected in the prefrontal cortex and the motor cortex, which are located at the downstream of primary associative memory cells29–31.\n\nTo investigate the formation and working principle of associative memory cells, we need more complete animal models for associative memory, which can match the features of associative memory in human beings. Associative memory in humans comprises of a signal inducing the recall of its associated signals, or the other way around, for logical reasoning, associative thinking and imagination in forward and backward manners. In animals, fear conditioning, eyelid-blinking conditioning and operant conditioning are used to examine cellular and molecular mechanisms underlying associative memory32–35. After eyelid-blinking conditioning or fear conditioning based on sound signal is established, whether air-puffing to the cornea or electrical shock to the feet induces the recall of the sound signal remains unknown. In addition, the electrical shock may activate entire sensory cortices and even the whole brain by spreading electrical current in the body, so that the association is not region-specific in the brain. Current studies indicate that the associations of whisker and olfactory stimulations in mice lead to odorant-induced whisker motion and whisker-induced olfactory response2,12–14, a comprehensive model to investigate associative memory. In this review, we aim to summarize associative memory cells and their working principles.\n\n\nCellular changes in associative memory\n\nBased on studies with animal models including fear conditioning, eyelid-blinking conditioning and operant conditioning in rodents32–36 and withdrawal reflex in Aplysia37–39, the following mechanisms may be involved in associative memory. From a psychological perspective, a conditioned signal induces the prediction of an unconditioned signal forthcoming as the basis of a conditioned reflex; however, the neural substrates remain unclear4. In terms of memory location, the movement-related brain areas and motor neurons1,40–42, or the sensory cortices43,44 presumably encode the storage of those associated signals; however, the characteristics and working principle of these neurons that are working in coordinated manner for memory formation remain to be elucidated2,3. In terms of the functional status of synapses and neurons, activity-dependent plasticity, e.g., long-term potentiation and depression of synaptic transmission45,46, is presumably involved. How these types of neuronal plasticity in a given pathway are correlated with the integration and storage of these associated signals has not been addressed. In addition, perceptual memory presumably resides in the cell assemblies formed by the strengthening of neuronal connections due to their correlated activities in information acquisition47. However, the natures of these cell assemblies, the patterns of neuronal connection strengthening, and the correlation between cell assemblies and their strengthening are largely unknown.\n\nObviously, the current knowledge as indicated above does not inform us of the neural substrates for integrative storage of associated signals or their working principles. Neural plasticity alone cannot account for memory patterns, such as explicit versus implicit memory, declarative versus non-declarative memory, episodic versus semantic memory and transformation between such patterns48, as well as the temporal features of memory from sensory inputs toward short-term and long-term memory. How memory constitutes the foundation of cognitive processes, such as associative thinking, logical reasoning and so on, remains unknown. How endogenous signals generated from logical reasoning and associative thinking is memorized for future presentation remains unknown. How memory is encoded under the different consciousness states is largely unclear. Thus, a comprehensive understanding of cellular mechanisms underlying associative memory should be established like an effort to see individual tress and the forests as well.\n\n\nAssociative memory cells in sensory cortices\n\nAssociative memory cells are presumably recruited for the integrative storage of associated signals49. The association of sensory signals leads to their integrative storage and retrieval, so that each signal evokes the recall of its associated signals. In terms of cellular substrate, the co-activation of sensory cortices induces mutual synapse innervations among these cortices, and recruits associative memory cells to integrate and encode these associated signals2,13,14.\n\nIn the study of this assumption, associative learning by paring whisker and odor stimulations in mice leads to odorant-induced whisker motion and whisker-induced olfaction response. Their barrel cortical neurons are able to encode new odor signals and innate whisker signals, as well as receive new synapse innervations from the piriform cortex alongside innate ones from the thalamus. Their piriform cortical neurons encode new whisker signals and innate odor signals, as well as receive new synapse innervations from the barrel cortex alongside innate ones from the olfactory bulb. In other words, a portion of barrel and piriform cortical neurons in mice that express this associative memory receives new synaptic inputs based on their mutual innervations in conjunction with innate synaptic inputs, so that these neurons encode associated new and innate signals, i.e., associative memory cells2,10,13,14. The neurons that encode either one of these signals are called as new memory cells or innate memory cells2. These associative memory cells include glutamatergic neurons, GABAergic neurons and astrocytes2,10,12,15. Moreover, associative memory cells can encode more than two signals9,50,51. Paired whisker, odor and tail stimulations lead to odorant-induced and tail-induced whisker motions alongside whisker-induced whisker motion. Associative memory cells in correspondent sensory cortices are recruited to encode three associated signals via their mutual synapse innervations (9,51 and Figure 1). Therefore, associative memory cells and mutual synapse innervations among sensory cortices constitute cellular substrates to memorize specific associated signals, in which the co-activation and simultaneous activity of these cortices are essential for new synaptogenesis and associative memory cell formation. As there are no natural connections among these cortices in mice, this associative learning model can be considered as “artificial” intelligence in animals.\n\nTop includes three groups of primary associative memory cells (green, red and blue) in the sensory cortices. There are mutual innervations among associative memory cells in each group (intramodal) and among three groups of associative memory cells (cross-modal). Bottom demonstrates secondary associative memory cells (orange) in cognition- and emotion-related brain area, and where these cells are mutually innervated. The axons of primary associative memory cells project to secondary associative memory cells whose axons project back to primary associative memory cells. All of neurons possess innate synapse innervations (yellow axons).\n\nIn terms of molecular mechanisms for the recruitment of associative memory cells, some microRNA, such as microRNA-324 and microRNA-133, levels increase12,15. Associative memory, new synaptic innervations and associative memory cells are attenuated by antagomirs for microRNA-324 and microRNA-133a9,52. Therefore, microRNA-mediated epigenetic processes, through lowering ttbk1 and tet3 expression, appears to be involved9,51,52. The blockade of ttbk1 and tet3 can weaken the limiting factor for the axon prolongation and synapse function53,54. Therefore, the formation of new synapse innervations and the recruitment of associative memory cells may be based on a chain reaction of intensive spikes, microRNA expression changes and epigenetic-regulated genes and proteins that manage axon prolongation and synapse formation9,10,52.\n\nWhere is the primary location of integration of associated signals? Inhibiting the function of sensory cortices blocks reciprocal cross-modal reflexes2,10. Applying microRNA antagomirs to these sensory cortices significantly weakens the strength of associative memory as well as the recruitment of new synaptic innervations and neurons that encode associated signals9,52. Therefore, the primary areas to encode associative memory are likely located in sensory cortices, where mutual synapse innervations and associative memory cells, called as primary associative memory cells, are recruited after associative learning3. It is worth noting that the association of cross-modal sensory signals may occur in visual versus auditory signals, auditory versus olfactory signals, auditory versus taste signals, and auditory versus somatosensory signals, such that primary associative memory cells can be recruited in visual, auditory, olfactory, gustatory and somatosensory cortices through their mutual synapse innervations (3 and Figure 2 and Figure 3).\n\nThere are four stages of associative learning and memory, associated signals’ acquisition, exogenous information integration and storage, endogenous information integration and storage as well as information retrieval by behavioral presentation. Associative memory cells (AMC) are classified into primary AMCs in sensory cortices that integrate and memorize exogenous information and secondary AMCs in cognition-, emotion- and behavior-related brain areas that memorize endogenous information during associated cognition and emotion events. Cross-modal associative memory cells are recruited by mutual innervations among sensory cortices or between cognition- and emotion-relevant brain regions. Intramodal associative memory cells are recruited by mutual innervations among the neurons in a single-modality sensory cortex, cognition brain area or emotion brain area. In addition to activations by innate input and new mutual innervations from co-activated brain regions to integrate and encode associated signals, associative memory cells are activated by the arousal system including the ascending reticular activating pathway in the brain stem and thalamus as well as the ascending activating pathways from the cholinergic nuclei, midbrain raphe nuclei and locus coeruleus that release acetylcholine (ACh), serotonin (5-HT) and norepinephrine (NE), respectively, which can maintain well wakefulness, permit normal consciousness as well as grant specific alertness and attention. Primary and secondary associative memory cells innervate and activate memory-output cells in the motor cortex for memory presentations by languages, gestures and countenance. Emotional reactions are often accompanied by the activities of autonomic nerves and hypothalamic hormones. Emotion-related brain areas include the amygdala, nucleus accumbens (NAc), ventral tegmental area (VTA) and so on that are involved in emotional reactions. Cognition-related brain areas, such as the prefrontal cortex, work for cognitive processes. The upregulations of AMC number and activity strength can facilitate memory to be impressive, or vice versa. The functional downregulation of motion-related brain areas leads to the inability of memory retrieval and presentation.\n\nA) Learning apple’s image and word “APPLE” composed by letters associatively. Intramodal associative memory cells (red) in the visual cortex (VC) are recruited through their mutual innervations to encode apple image about different features. Intramodal associative memory cells (blue) in auditory cortex (AC) are recruited through their mutual innervations to encode word “APPLE” based on their composed letters by listening the sound of letters and word. The association of apple’s image and APPLE leads to the co-activation of associative memory cells in the visual cortex and auditory cortex, so that mutual synapse innervations between these two groups of associative memory cells as well as cross-modal associative memory cells are recruited. B) Learning orange’s image and word “ORANGE” composed with letters associatively. Intramodal associative memory cells (orange) in the visual cortex (VC) are recruited by their mutual innervations to encode orange image about different features. Intramodal associative memory cells (cyan) in auditory cortex (AC) are recruited by through their mutual innervations to encode word “ORANGE” based on their composed letters by listening the sound of letters and word. The association of orange’s image and ORANGE leads to the co-activation of associative memory cells in the visual cortex and auditory cortex, such that mutual synapse innervations between these two groups of associative memory cells as well as cross-modal associative memory cells are recruited. C) Learning banana’s image and word “BANANA” composed with letters associatively. Intramodal associative memory cells (green) in the visual cortex (VC) are recruited through their mutual innervations to encode banana image about different features. Intramodal associative memory cells (green) in the auditory cortex (AC) are recruited by their mutual innervations to encode word “BANANA” based on their composed letters by listening the sound of letters and word. The association of orange’s image and BANANA leads to the co-activation of associative memory cells in the visual cortex and auditory cortex, so that mutual synapse innervations between these two groups of associative memory cells as well as cross-modal associative memory cells are recruited. D) Associatively learning images (apple, orange and banana) and words “APPLE, ORANGE and BANANA” coactivates associative memory cells in the visual cortex for these images as well as associative memory cells in the auditory cortex for these words. These groups of cross-modal associative memory cells are mutually innervated, and their coactivity upregulate their functional state. These functionally regulated associative memory cells are easily activated by the cues that lead to dominant recall and memory presentation, in which the coactivity-dependent cycle in the refinement and recruitment of associative memory cells is involved. The functionally upregulated cells for encoding images are labeled by dark red, orange and green color, as well as those for encoding words are labeled by dark blue, in comparison with those before their associative coactivation. Cells include ready-recruited neurons and naïve neurons are labeled by yellow. All of the neurons receive their innate input (yellow axons).\n\nTaking these studies together, we propose the characteristics of associative memory cells in sensory cortices. The co-activation and simultaneous activity of cortical neurons are essential to generate new synaptic innervations and recruiting associative memory cells. They receive new synapse innervations from co-activated sensory cortices for their mutual connections as well as innate sensory inputs. They encode new and innate associated signals for their integrative storage. Their axons project to cognition- emotion- and behavior-related brain regions for cognitive event and memory presentation. The number of associative memory cells and their upregulated refinement influence memory strength and maintenance. The activation of associative memory cells permits logical reasoning, associative thinking, and so on. Their recruitment is influenced by epigenetic-regulated genes and proteins that manage axon prolongation and synapse formation9,10,52. In the integration, storage and retrieval of associated signals, the working principle of associative memory cells is based on their reception strength to innate and new synapse inputs, their ability to convert synaptic analogue signals into digital spikes for encoding associated signals and their ability to output sequential spikes55–58 that will drive behavior-, cognition- and emotion-related brain areas. Thus, synapse innervations to associative memory cells determine the specificity of memory contents. The number, activity level and plasticity of associative memory cells as well as the connection and activity strengths in their input and output partners set up the power and persistence of information storage and memory presentation9,11,52,59. For instance, barrel cortical neurons receive new synapse inputs from the piriform cortex after associative learning in conjunction with innate inputs from the thalamus. Synapse activities induced by odor stimulus will drive barrel cortical neurons toward the threshold of firing spikes under the condition of basal thalamic input, and their spikes then activate motor cortical neurons for odorant-induced whisker motion. With associative memory cells in sensory cortices2,9,10,15,52, their axon-innervated downstream neurons are able to encode these associated signals16,21,25,26,29–31. The stimulation to any of these areas in neural circuits from sensory cortices to behavior- and emotion-related brain nuclei can induce memory presentation19,20,23,24,27,28.\n\n\nAssociative memory cells in cognition-, emotion- and behavior-related brain areas\n\nIn addition to primary associative memory cells in sensory cortices for the integration of exogenous signals, secondary associative memory cells that work for the integrative storage of endogenous signals may be recruited in cognition, emotion and behaviors3. This assumption is based on the following facts in life. The content, process, and consequence of logical reasoning and associative thinking can usually be remembered and stated. For instance, we often can tell that images are from previous sights, words from previous reading or listening, tastes from previous eating, and so on. Emotional reactions to various stimulations and operation processes can be recalled for subsequent description. These specific events in the mind may be generated based on the associative storage of previous exogenous signals in sensory cortices, and memorized in brain regions relevant to cognition, emotion or behaviors in an integrative manner. In terms of cellular substrates, the hypothesis is that the association of previously stored associative signals in sensory cortices makes primary associative memory cells strengthening their mutual synapse innervations, convergently innervating downstream neurons and even receiving synapse innervation back. These downstream neurons become to encode associative signals and are recruited as secondary associative memory cells that memorize specific contents in associative thinking and logical reasoning, and their interactions form associative thinking and logical reasoning with the inclusion of sensory origins3.\n\nCertain studies have been conducted to examine the involvement of brain areas including prefrontal cortex, hippocampus and amygdala in memory formation24,60. Neurons in the medial prefrontal cortex demonstrate a sustained activity after paired stimuli25,26. Cue-selective neurons are recorded in the inferotemporal cortex after pair association memory21. Neurons in response to conditioned and unconditioned stimuli and their response transformation are detected in the amygdala61. Neurons in the hippocampus and amygdala take part in contextual fear memory62. Memory assemblies for temporal information are overlapped and recorded in the hippocampus16. The activation of engram cells in the amygdala or hippocampus is sufficient to induce fear responses19,20. This indicates that memory cells are generated in the prefrontal cortex, hippocampus and amygdala for memory retrieval. However, whether these memory cells emerge and are innervated secondarily by associative memory cells in sensory cortices remains to be examined.\n\nIn a mouse model of associative learning by pairing whisker and odor signals, neurons that encode whisker and odor signals are detected in the motor cortex and prefrontal cortex30,31, in addition to barrel and piriform cortices2,12. Their responses are reduced by inhibiting the activity of barrel or piriform cortical neurons. Their responses and plasticity are sustained in the barrel cortex long-term, and decayed in the motor cortex after pair training ends. Moreover, individual neurons in the prefrontal cortex and motor cortex receive synaptic innervations from barrel and piriform cortices after pairing odor and whisker stimulations29–31. This provides functional and morphological evidence for the recruitment of secondary associative memory cells in the prefrontal cortex, motor cortex and so on, based on the co-activation of neurons in these areas with primary associative memory cells (3 and Figure 2).\n\nCurrent studies appear to imply that secondary associative memory cells in different brain areas undergo cross-modal connection, similar to primary associative memory cells among sensory cortices3. For instance, the pathway from the ventral hippocampus toward the nucleus accumbens is involved in social memory22. Engram cells in the prefrontal cortex emerge after receiving the inputs from the hippocampus and amygdala during contextual fear memory18. The axon projection from the prefrontal cortex and hippocampus to the amygdala is formed during fear memory63. The pathway from the prefrontal cortex to the striatum plays a crucial role in reward memory23.\n\nBased on these studies, the characteristics of secondary associative memory cells are proposed below. They receive new synapse innervations convergently from primary associative memory cells in those co-activated sensory cortices in cognition and emotion events. They encode endogenous associated signals for their integrative storage in cognition and emotion. The association of cognition process and emotion reaction evokes mutual innervation among secondary associative memory cells in cognition- and emotion-related brain areas. Their axons project to memory-output cells in behavior-related brain areas for memory presentation by language, countenance, gesture and writing. The number of recruited secondary associative memory cells and their upregulated refinement influence the memory strength and maintenance of cognitive and emotional contents. The activation of secondary associative memory cells permits the rehearsal of associative thinking, logical reasoning, emotional reactions and so on. Working principle for secondary associative memory cells is based on their reception of synaptic inputs, their capability to convert synaptic analogue signals into digital spikes for encoding associated signals, as well as their ability to output spikes that drive memory-output cells. Thus, synapse innervations to secondary associative memory cells determine the specificity of memory contents in cognition and emotion. The number, excitability and plasticity of secondary associative memory cells, as well as their connection and activity strengths, set up the persistence and power of information storage and memory presentation. It is pointed out that the outputs of secondary associative memory cells innervate brain areas, such as the hypothalamus and extrapyramidal system, to affect sympathetic/parasympathetic balance, temperature setpoint, food ingestion and hormones to be involved in emotional reactions and behaviors.\n\nAssociative memory cells can be recruited among cross-modal sensory cortices or their downstream brain areas related to cognition and emotion through mutual synapse innervations. Moreover, associative memory cells may be recruited based on their mutual innervations in intramodal brain areas, such as associated images to the visual cortex64, associated odors to the olfactory cortex, associated words to the auditory cortex and so on (Figure 1–Figure 3). Associated signals from a given sensory input to its correspondent sensory cortex may innervate the different groups of neurons. The coactivation of these groups of neurons induces their mutual synapse innervations, so that associative memory cells in a single modality of the sensory cortex are recruited, which memorize intramodal signals with different features, strengths and locations of input signals. These associative memory cells in intramodal sensory cortices fulfill the intramodal memory of these associated signals, such that image one induces image two recall, odor one induces odor two recall and word one induces word two recall, or the other way around (Figure 1–Figure 3)3. In fact, there is usually a time delay among intramodal signals. The activity persistence in the different groups of neurons in a given sensory cortex influences whether their coactivations overlap to recruit intramodal associative memory cells. The different portions, activity levels and connections of these recruited associative memory cells may determine the storage and retrieval of intramodal signals in different features65. In addition, intramodal associative memory cells may be recruited in brain areas related to cognitive events and emotional reactions3.\n\n\nPlasticity in associative memory cells\n\nCell assemblies formed by the strengthening of neuronal connection due to their correlated activities may be involved in information acquisition, especially the coincidence of activity of presynaptic and postsynaptic cells47. This hypothesis has led neuroscientists to study the involvement of synaptic and neuronal plasticity in memory formation for several decades. Plasticity in synapse connection and neuron activity is presumably the cellular mechanisms underlying learning memory5,6,66,67, such as long-term potentiation and depression in synaptic transmission45,46 or neuronal activity26,68. However, some points have been ignored in these studies. Synaptic and neuronal plasticity is examined in brain areas presumably related to memory formation, but not in memory cells. Synaptic plasticity in a given neural pathway does not reveal how multiple signals are integrated and stored in associative memory cells. These uncertainties raise an issue of how these data will be integrated into cellular mechanisms underlying associative memory.\n\nIn terms of synaptic plasticity on associative memory cell, the number of excitatory synaptic inputs and the strength of individual synapses on glutamatergic and GABAergic neurons are upregulated, glutamatergic neuron’s outputs are upregulated as well as GABAergic neuron’s outputs are downregulated12,15,29,59,69. In neuronal plasticity, primary and secondary associative memory cells in vivo express activity-dependent upregulation in their active population and activity strength in response to associated signals31, the intrinsic property of glutamatergic associative memory cells is upregulated and the excitability of GABAergic associative memory cells is downregulated12,15,59,69. Mutual innervation among associative memory cells is upregulated12,15. These factors coordinately shift the balance between excitation and inhibition to more excitation at cortical neurons that may be the driving force in recruiting more mutual synapse innervations as well as glutamatergic and GABAergic associative memory cells, to promote their functional state to an optimal level for storing associated signals, and to facilitate the activation of associative memory cells for the recall and presentation of associated signals11. In associative memory cells with new synapse innervations, their upregulated excitatory input and downregulated inhibitory input can increase their active states to the higher level for receiving and storing new information, i.e., the recruitment of more associative memory cells2,10,15. The raised number and function of excitatory synapse inputs strengthen the encoding ability and precision of associative memory cells for information storage and precise and efficient retrieval55–57. If excitatory associative memory cells are overly active, they activate the neighboring inhibitory neurons to prevent them from the hyperactivity through recurrent negative feedback55,70–72.\n\nThere are two forms of neuronal excitation plasticity to interpret how neuronal plasticity is involved in the formation and the retrieval of associative memory, i.e., the downregulation of threshold potential to fire spikes and the upregulation of spiking ability to fire more spikes. It has been found that the intensive activity of the neurons by high frequency stimulation, similar to neuronal activation by inputting learnt signals, decreases neuronal threshold potential close to the resting membrane potential, so that the firing of neuronal spikes is facilitated68. Their plasticity in multiple grades68 allows associative memory cells to handle the different groups of associated signals. Furthermore, intensive neuronal activity upregulates cell capacity to fire sequential spikes26,67. Both mechanisms enhance neuronal capability to encode digital spikes for the recruitment of new synaptic innervations and associative memory cells as well as the retrieval of stored associative signals. These features have been observed in associative memory cells12,15,59. These data indicate that the plasticity of neuronal excitability may also play a central role in learning and memory, which is reiterated by a current review73.\n\nBased on the discussion above, we summarize the following points for the integration and storage of associated signals. The formations of primary associative memory cells in sensory cortices and of secondary associative memory cells in cognition/emotion-related brain areas endorse the specificity of the stored associative signals2,3,9,10,12,15,52. The number and functional state of associative memory cells influence the strength and maintenance of information storage as well as the recall and presentation of the stored information9–11,52. Structural and functional plasticity at subcellular compartments of associative memory cells determines whether they sensitively integrate associated signals, precisely memorize these signals, and efficiently trigger neurons in their downstream brain areas for memory presentation12,15. The recruitment of associative memory cells by new synapse innervations and the plasticity of their function states are critical for information storage and retrieval. In addition, both recruitment and refinement of associative memory cells depend upon the simultaneous activity of neurons2,9,10,12,15,52. The activities of associative memory cells as a central point comprise coactivity-dependent cycle in their recruitment and refinement, i.e., activity together, wiring together and strengthening together. Highly active neurons in learning associated signals recruit associative memory cells and upregulate their functions. The upregulated population and function state of associative memory cells in these repeated learning events recruit more associative memory cells and upregulate their functions further. This coactivity-dependent positive cycle that is based on functional compatibility between neuronal partners58 can interpret realistic practices under the condition of normal consciousness and high attention, that is, the more learning times is, the more associative memory cell recruitment and refinement is, and the more impressive memory is. It is noteworthy that associative memory cells fall into the active group of neurons in the brain, but non-specific c-fos-labelled active cells in the brain may not be memory engrams.\n\n\nWorking principles of associative memory cells\n\nAssociative memory cells are essential for memory formation and related cognitions2,9,29–31,52,59,69. If it is true, their natures and working principle can also be used to interpret the processes of associative learning and memory, such as the efficiency of associative learning, the integrative storage of specifically associated signals, the strength and maintenance of associative memory, the efficiency of memory retrieval, the transformation of simple to complicated information storage, the correlation of associating memory to cognitive process and emotional reactions, and so on. Moreover, memory patterns, such as explicit versus implicit memory, declarative versus non-declarative memory, episodic versus semantic memory as well as transformation between these patterns, remain to be figured out in cellular bases. How associative memory is encoded under the different states of consciousness and attention remains unknown. Here, we discuss the working principles of associative memory cells in these aspects of learning and memory.\n\nThe simultaneous activity of neurons in different brain regions is essential for the recruitment of associative memory cells. The co-activation of sensory cortices induces their mutual synapse innervations, such that associative memory cells will be recruited2,9,12,15. The axons of these primary associative memory cells convergently innervate the cognition/emotion-related brain areas to recruit secondary associative memory cells in logical reasoning and associative thinking29–31. These populations of associative memory cells, based on their received synapse inputs among co-activated brain areas, constitute the memory specificity of associated signals. The coactivity-dependent positive cycle in the recruitment and refinement of associative memory cells promotes the strength and maintenance of associative memory. These results advance a classical hypothesis that the groups of repeatedly co-activated cells become wired and that the strengthening of neuronal wiring forms cell assembles for memory47.\n\nIn terms of the driving force to activate and maintain the activities of neurons and associative memory cells, there are three resources, including new synaptic innervations from co-activated brain regions, innate synaptic inputs formed during development, as well as synaptic inputs from non-specific ascending pathways in the arousal system. The ascending reticular activating pathway from the brain stem and the thalamus receives various sensory inputs and widely innervates the cerebral brain to allow wakefulness and consciousness74–76. The ascending pathways from neuronal axons in the cholinergic nuclei, midbrain raphe nuclei and locus coeruleus innervate the forebrain to maintain alertness and consciousness by releasing acetylcholine, serotonin and norepinephrine77–79. With this arousal system to uphold the basal activation of associative memory cells, they are able to integrate innate and new synaptic inputs specifically and to memorize these associated signals. Similarly, this arousal system may influence the efficiencies of associative learning and memory retrieval as well as the association of memory with the cognitive process and emotional reaction.\n\nThe efficiency of associative learning is affected by the intrinsic property of neurons, their responsiveness to synaptic inputs, and the number of neurons ready to be recruited. Neurons ready to be associative memory cells may have stored signals relevant to the topic that will be learnt, and can be activated by giving topic cues in attention call. The proportion of recruitment-ready neurons influences how the information is acquired and memorized easily as well as how the complicated signals are efficiently learnt (please see below). This is one reason why the efficiency of associative learning is influenced by whether individuals are knowledgeable in the topic to be learnt. In addition, cortical neurons are diverse in their synapse input and intrinsic property55. Neurons with more synaptic inputs and lower threshold potential are easily activated to fire spikes for high learning efficiency2,59, which triggers a chain reaction of intensive spikes and microRNA expression changes for axon prolongation and synapse innervations9,52. Thus, activity-dependent upregulation in neuronal excitability and synapse innervations facilitates the learning efficiency.\n\nThe efficiency of information recall and memory presentation is affected by the number and functional state of associative memory cells as well as the coactivity-dependent cycle of associative memory cell recruitment and refinement. In general, the recruited number of associative memory cells is proportional to the activated associative memory cells in memory retrieval under the condition of normal consciousness and alertness, so that the efficiency of memory retrieval would be consistent to the efficiency of associative learning2,59. The functional state of associative memory cells influences how they are easily activated during memory retrieval15. As discussed above, the coactivity-dependent cycle in the recruitment and refinement of associative memory cell will add more associative memory cells into the memory-unit pool in information storage areas, such that the efficiency of memory retrieval would be higher under the condition of normal consciousness and alertness. In addition, whether the stored information can be successfully retrieved is dependent on the functional state of memory-output cells, since the functional downregulation of memory execution cells in the motor cortex leads to the inability of memory retrieval (i.e., memory extinction) though primary associative memory cells are well-maintained in their normal function11,30.\n\nIn the transformation from exogenous signals to endogenous signals and their integrative storages3,29–31, the efficiency to correlate associative memory with cognitive processes and emotional reactions is a critical issue. In this process, the interaction between primary and secondary associative memory cells based on their mutual synapse innervation (Figure 1), as well as the number, function state and plasticity of these associative memory cells, should be accounted during logical reasoning and associative thinking. Thus, cellular processes involved in the efficiency for the learning, storage and retrieval of exogenous associated signals may similarly work for the transformation of exogenous-to-endogenous signals.\n\nAssociative learning and memory in life involves a gradual process where individuals memorize associated signals from simple to complication, i.e., the transformation of simple to complicated information storage, in a topic-related manner3. Initially, simple images with different intramodal features and words based on letters are jointly learnt to activate neurons in visual and auditory cortices. With their mutual synapse innervation, associative memory cells (AMC) are recruited in intramodal and cross-modal manners including AMCs for pictures (AMCPP), for letters (AMCLL) as well as for pictures and words (AMCPL). Their plasticity and reactivation will recruit more associative memory cells to initiate the coactivity-dependent cycle in recruitment and refinement, i.e., the first grade of associative memory cells. Through the accumulation of associative memory cells that store simple images and words, they are ready-recruited neurons that become to encode complicated associative signals during learning complicated images and sentences. Subsequently, the complicated images and sentences are associatively learnt to activate the first grade of associative memory cells in visual and auditory cortices. Through their mutual synapse innervation, the second grade of associative memory cells to encode complicated images and sentences are recruited including associative memory cells for complicated pictures (AMCCPP), for sentences (organized words, AMCOWW) and for pictures and sentences (AMCPPS). Through these processes, numerous groups of the first and second grades of associative memory cells are recruited and accumulated in life-time learning. In the advanced learning, multiple grades of associative memory cells are recruited to encode more complicated signals. Once the different groups and grades of associative memory cells are recruited, the subsequent learning and memory will initiate plasticity at these associative memory cells based on their intensive activity31, which become easily activated for quick learning and memory. Reading books or looking at images induces intense activity in certain groups of associative memory cells that encode sentences and images, which leads to activity-dependent upregulation at these associative memory cells. Their low threshold potential to fire spikes68, and active synapse inputs to drive these cells will permit the cues dominantly to activate them for recall of images and sentences, and even the spontaneous activation of these cells to drive secondary associative memory cells for free associative thinking. These associative memory cells will lead to memory presentation by behavior if they successfully drive the activation of memory-output neurons in the motor cortex. It is noteworthy that the complicated signals can also be dissected and memorized through the formation of associative memory cells that are able to encode two signals, three signals, and even more signals9. The partial activations of these associative memory cells lead to the selective recall of these complicated signals.\n\nIn terms of the correlation of associative memory cells to memory patterns, such as explicit versus implicit memory, declarative versus non-declarative memory, episodic versus semantic memory and transformation between these patterns, our views are given below. Although these are psychological classifications, there is no clear border line to separate them. Explicit or declarative memory is intentional recall consciously, and implicit or nondeclarative memory is effortless recall without conscious awareness. In fact, the formation of so-called implicit memory has been implicated when individuals initially learn these processes and operations. With long-term practice to be skilled, the recall and expression of these processes and operations no longer require conscious effort. As a result of the coactivity-dependent cycle between the recruitment and refinement of associative memory cells, the repeated activity of primary and secondary associative memory cells will recruit more associative memory cells and upregulate their functions2,12,15, as well as strengthen synapse connections from associative memory cells to memory-output cells in the motor cortex11,30, so that explicit memory can be converted into implicit memory. In other words, there may be a negative relationship between the number and upregulation of associative memory cells and the requirement of consciousness, a homeostasis for memory retrieval. Implicit memory is based on more associative memory cells that are easily activated, supported by phenomena that implicit memory can usually be expressed spontaneously. In explicit memory, episodic memory in individual events can be converted to semantic memory. In general, once associative thinking and logical reasoning with their repetitions place all associative memory cells that store the events with similar topics together to reorganize them into a group of memory cells for general concepts and/or to convergently innervate on another grade of associative memory cells in an abstract manner.\n\nConsciousness is the combinational state of wakefulness and memory, where individuals are aware of and identify themselves and objects in the environment80, which may be based on the basal activation of associative memory cells by the ascending arousal system and the specific activation of associative memory cells by their associated inputs through sensory cues. In this regard, the number and functional state of associative memory cells is proportional to the state of consciousness. The combination of consciousness and a specific alert constitutes attention, i.e., a specific group of associative memory cells are activated. Once individuals are under consciousness, they possess two forms of logical reasoning and associative thinking, i.e., critical versus creative. Critical thinking activates more recruited secondary associative memory cells for evaluation, whereas creative thinking may generate newer secondary associative memory cells for inspiration.\n\nThe awareness state can be classified into consciousness and unconsciousness. Sleep can fall into unconsciousness (slow wave sleeping) and incomplete consciousness (fast wave sleeping)80. How do different groups of associative memory cells work together during fast wave sleeping or dreaming? Dreams are often accompanied by high activities in electronic encephalograph and behaviors, such as rapid eye movement, muscle twitch and active respiration/heat beat, indicating high activity in the forebrain. In the meantime, associative memory cells in the large population for specific images and events are presumably activated, especially those cells intensively activated and frequently thought during daytime, such that these images and events are played back. Because of the negative relationship between the number and upregulation of associative memory cells, and the requirement of consciousness, a large portion of associative memory cells can be activated under partial consciousness condition, which makes the playback of images and events being incomplete duplicates of realistic ones. Because playbacks can be recalled and described, associative thinking and logical reasoning (the integration of endogenous signals) based on primary and secondary associative memory cells can be fulfilled under incomplete consciousness3.\n\nThe signals from different sensory modalities and various events can be expressed by word-based language during associative thinking and logical reasoning. The associations of these sensations or events with their word descriptions occur during initial learning, where the co-activation of these cortical areas recruits associative memory cells that encode these sensations/events and word descriptions. While the sensations and behaviors are recalled in sequential playbacks, associative memory cells for their word descriptions are triggered, such that word descriptions substitute the complicated images and events to speed up these cognitive processes3. The substitution of words to images is realized based on the recruitment of more associative memory cells and their upregulation in coactivity-dependent cycle manner by repeated practices. However, if words and sensation/events are associated improperly, the correction of these associations is difficult because of the presence of these recruited synapse innervations, associative memory cells and their circuits.\n\n\nModulation of associative memory cells\n\nIn addition to specific new synapse innervations and innate inputs, there is no substantial evidence indicating the modulation of associative memory cells by other synaptic inputs and neurotransmitters. The arousal system including the ascending reticular activating pathway74,75 and ascending activating pathways from the neuronal axons of the locus coeruleus, midbrain raphe nuclei and cholinergic nuclei77–79,81 widely innervates the forebrain to maintain wakefulness and to permit consciousness. Their released neurotransmitters, acetylcholine, serotonin and norepinephrine, theoretically modulate the functional states of ready-recruited neurons and associative memory cells. The activity of this arousal system maintains the basal activation of associative memory cells that integrate new and innate synaptic inputs, as well as memorize these associated signals specifically. In other words, the alert and reward may facilitate the recruitment and refinement of associative memory cells.\n\nThe modulation of learning memory by acetylcholine, norepinephrine and serotonin has been documented82–85. The direct activation of acetylcholine M1-type receptors on hippocampal interneurons contributes to learning memory86. The infusion of norepinephrine or adrenoceptor agonist into the amygdala or the prefrontal cortex enhances memory formation, which coordinates with the action of stress hormone87. The augmented and reduced activities of serotonin neurons lead to bidirectional influence on memory and cognition88,89. The action of dopamine onto type-I and type-V receptors in the forebrain and the hippocampus plays an important role in spatial learning and memory90,91. These data provide the evidence for neurotransmitters, such as acetylcholine, serotonin, norepinephrine and dopamine, as well as stress hormone to modulate learning and memory. How these neurotransmitters act onto the presynaptic inputs of associative memory cells to influence transmitter release or the bodies of associative memory cells to influence their excitability remains to be addressed.\n\nIt has been found that serotonin facilitates the neuron excitability and neuron responses to synaptic inputs92,93, and that the activation of dopaminergic neurons facilitates synaptic bouton formation and postsynaptic neuron activity in their target regions94. The plasticity of these monoaminergic neurons may modulate the recruitment and refinement of associative memory cells, and in turn influence memory formation and memory-related cognitions. This modulation supports the fact that the high levels of wakefulness, consciousness, attention and motivation based on the active monoaminergic and cholinergic neurons elevate the efficiencies of associative learning and memory retrieval.\n\n\nThe impact of associative memory cells on physiology and pathology\n\nThe memory of associated signals is important for establishing bidirectional alertness and prediction in life. With associative memory cells based on primary and secondary locations, as well as grade one, two or more integrations, one signal induces the recall of its associated signals and the expression of their respective behavior, or the other way around, such that individuals are able to fulfill logical reasoning and associative thinking, as well as to predict future events in a forward and backward manner. Moreover, associative memory cells in each of co-activated brain areas encode the associated innate signal and newly learnt signal. Each of associated signals is memorized at multiple brain areas, which largely reduces the chance of memory loss2,3. The storage of multiple signals in an associative memory cell strengthens the efficiency of memory retrieval9. The storage of multiple signals in a cortical area and the recall of one signal triggered by multiple signals enable these individuals to strengthen their abilities in memory retrieval and well-organized cognitions3.\n\nIt is widely accepted that the normal consciousness and attentiveness are important for memory formation48,95,96, which can be explained by associative memory cells and their characteristics. With the arousal system to maintain wakefulness and the activation of recruitment-ready neurons by giving topic cue in attention call, their activation and activity allow them to encode associated signals. These recruited associative memory cells under the wakefulness condition allow for individuals to identify themselves and objects in their environment, which constitutes consciousness. The consciousness supports the activation and activity of associative memory cells to enter the activity-dependent positive cycle in their refinement and recruitment, such that more associative memory cells are recruited and impressive memory is generated in mind.\n\nThe age-related change in the efficiency of learning and memory throughout life is a well-known phenomenon97,98. There is an uprising-peak-decline process of associative learning and memory2. In terms of cellular mechanisms, synaptic potentiation becomes matured during the postnatal development99, and neuronal excitability in mouse cortical neurons is upregulated to a plateau level at postnatal day 2268, which matches well with dynamic changes in associative memory2. The plasticity of neurons and synapses with the recruitment of associative memory cells in postnatal development initiates the coactivity-dependent cycle to recruit and refine associative memory cells, so that more associative memory cells are recruited to increase the efficiency of learning and memory59,69. In aged mammalians, the accumulations of β-amyloid and phosphorylated tau-protein in the brain may influence axon prolongation and synapse formation9,52 to block the recruitment of associative memory cells and/or to impair those recruited associative memory cells for memory deficits.\n\nIn terms of memory maintenance and extinction, the recruitment and plasticity of associative memory cells are not significantly decreased, but the activity of memory-output neurons in the motor cortex is lowered11,30. The sustained presence of associative memory cells as well as the recruitment of more associative memory cells during brain excitation confer associative memory to be recalled in lifetime, in which the information can be retrieved as long as their innervation onto memory-output neurons successfully drive them to be functionally active. It is noteworthy that memory recall shows different patterns in spontaneous, cue-induced and realistic object-triggered manner with the age. For instance, spontaneous recall occurs in child age and/or brain excitation, cue-induced recall usually occurs in the young and adults, and real object-triggered recall occurs in senior individuals. In addition, when the brain is highly excited in many regions, such as euphoria perception, extreme fear and strong stimulus, more associative memory cells are recruited in these areas through their mutual innervations, so that impressive memory and spontaneous recall to these experiences are generated in individual lifetime11,30. It is difficult to remove newly formed synapse innervations and recruited associative memory cells for the relief of fear memory. Alternative ways are the avoidance of fear stimulation and the induction of happiness to rebalance these two states to weaken fear memory, since the lack of uses in neural circuits related to fear memory3, especially from associative memory cells to memory-output neurons, may drive them to be functionally silent. In the brains of individuals with a history of substance abuse or addiction, primary and secondary associative memory cells related to these events are recruited in large amounts and in extensive areas under euphoria conditioning, leading to potential relapses in their lifetime3. Strategies for these individuals may include the avoidance of the environmental cues associated with substance abuse to weaken the output of the relevant associative memory cells, as well as the establishment of alternative sources of happiness to recruit associative memory cells, such that the rebalance of these two states will strengthen the memory-output pathway for happiness.\n\nThe proper upregulation of active neurons leads to their recruitment of associative memory cells, and the upregulation of associative memory cells facilitates the joint storage of associated signals3,15. These processes initiate the coactivity-dependent cycle of memory cell recruitment and refinement, so that more associative memory cells are recruited. However, the further upregulation of associative memory cells, e.g., the dysfunction of GABAergic neurons in schizophrenia and epilepsy100,101, allows associative memory cells to be overlaid and widely activated. The upregulated associative memory cells in sensory cortices will lead to hallucination, and the upregulated associative memory cells in cognition and emotion-related brain areas leads to delusion.\n\n\nConclusions\n\nAssociative memory cells are the basic unit to encode associated signals in objects and environments. Their recruitment and functional upregulation essentially determine the efficiency of learning and memorizing specifically associated signals. Their activation and persistent activity lead to the recall of memorized associated signals in the mind, and the presentation of stored signals by behaviors if they successfully activate memory-output cells. Morphological basis for these associative memory cells to encode associated signals is their receptions of innate input and new synapse innervations from co-activated brain areas. Based on the localization of associative memory cells, they are classified into primary groups to integrate exogenous signals in sensory cortices and to innervate neurons in cognition and emotion brain areas, as well as secondary groups to receive innervations from primary groups and to integrate endogenous signals during cognitive processes. Based on the complication of integrating associated signals, associative memory cells are classified into grade one, grade two and so on, whose activity-dependent upregulation works for the storage and retrieval of complicated signals. Associative memory cells with their upregulation lead to them being more active and recruit more ready-neurons to be associative memory cells, i.e., a coactivity-dependent cycle in the recruitment and refinement of associative memory cells for impressive memory in repetitive learnings. Associative memory cells are the basic unit for the storage of associated signals that influence the contents of cognitions and emotion reactions. The consequences and processes of cognition and emotion recruit more associative memory cells for them to be stored. The cycles in memory and cognition allow the experiences, capabilities and skills to be strengthened. In addition, the functional state of associative memory cells is modulated by the arousal system from the brain stem including the ascending reticular activating pathway and the ascending pathways from midbrain raphe nuclei, locus coeruleus, cholinergic nuclei and substantial nigra, which affect the efficiency of learning and memory. Working maps for associative memory cells are given in Figure 1–Figure 3.", "appendix": "Author contributions\n\n\n\nJW contributed to the concept, project design and paper's writing. SC contributed to drawing Figure 1 and Figure 3.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study is funded by National Key R&D Program of China (2016YFC1307100) and Natural Science Foundation China (81671071, 81471123) to JHW.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nIt is noteworthy that thousands and thousands of papers related to learning and memory have been published on public platforms. 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PubMed Abstract \| Publisher Full Text\n\nGalanopoulou AS: Mutations affecting GABAergic signaling in seizures and epilepsy. Pflugers Arch. 2010; 460(2): 505–23. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30314", "date": "05 Feb 2018", "name": "Ru-Rong Ji", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely review on an important topic, associative memory. The first author is a leader in this area. All the figures are well presented. Figure 1 is very informative. I only have a few minor concerns.\n\nIt will help if the authors can include a table to summarize key literature related to this topic.\n\nAre there any differences in cellular signaling mechanisms between primary and associated memory? If so, a table to summarize similarities and differences will be very useful.\n\nDo glial cells play a role in associative memory?\n\nNo pain, no gain? Is pain also associated with this type of memory?\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "30315", "date": "08 Feb 2018", "name": "Wen-Jun Gao", "expertise": [ "Reviewer Expertise Prefrontal cortex", "cognition", "electrophysiology", "and psychiatric disorders." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the present review, Wang and Cui have summarized their previous works about associative memory cells and made a hypothesis of how it works. Specifically, they named the cells that process exogenous associated signals as associative memory cells which could be classified as primary (located in sensory cortices) and secondary (located in cognition-, emotion-related brain areas). The authors also discussed the molecular changes and plasticity of the associative memory cells and raised an interesting hypothesis about working principles of these cells. Overall, this review is compelling, well-written, and timely needed for the field. However, there are some points that are not clear:\nThe authors raised several interesting questions in the manuscript. But it would be helpful for the authors to provide a perspective section to elucidate how these questions should be addressed at the end of the manuscript.\n\nFigure 1 and the related paragraph. The authors’ claim that mutual innervation is the major pattern of primary and secondary associative memory cells connection. This model is quite simple and easy to understand. However, are there any other patterns exist in the associative memory cells? For example, the neocortex is hypothesized to function as cortical column. Does the column model fit in the associative memory cells connection as well? The authors described the functional plasticity in associative memory cells. Do these cells also exhibit morphological changes? For example, what about the spine density or spine shape changes during the memory acquisition or storage? Page 3, In terms of molecular mechanisms, there are many molecular changes during the process. It is not clear why the authors point out microRNA, which is non-coding RNA. Page 6, the authors stated that individual neurons in the prefrontal cortex and motor cortex receive synaptic innervations from barrel cortices……This might be the case for motor cortex. To our knowledge, prefrontal cortex does not receive innervations from the barrel cortex directly.\nPage 6, reference 26 is not related to the prefrontal cortex.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] }, { "id": "31454", "date": "05 Mar 2018", "name": "Jie Wu", "expertise": [ "Reviewer Expertise Neuroscience", "drug addiction", "epilepsy" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAssociative memory cell is a new term to describe the special groups of brain neurons that play the critical role in information/signal acquisition, integration and storage of exogenous associated signals. In this review Dr. Wang has provided a comprehensive picture of these cells, their working principle and modulation. I think this article provides new insights into improved understanding of molecular/cellular mechanisms of cognition, emotion and behaviors. The manuscript is well written and the figures are nicely presented and make sense. I recommend publishing this paper in this journal. Following are some my questions and hopefully authors can appropriately address them in this article.\n\nWhat are the basic phenotypes of the “associative memory cells”, are they glutamatergic, GABAergic cholinergic, or others? Do these “associative memory cells” play the unit function such as acquisition, integration and storage of exogenous associated signals? Have these “associative memory cells” a special distribution in the brain? Do these “associative memory cells” form the specific circuits that are closely associated to the cognition and emotion? What changes of these “associative memory cells” in the diseases that exhibit cognitive deficits including Alzheimer’s disease, drug addiction, schizophrenia.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-108
https://f1000research.com/articles/7-106/v1	25 Jan 18	{ "type": "Research Article", "title": "Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells", "authors": [ "Novi Silvia Hardiany", "Purnamawati Huang", "Syarifah Dewi", "Reni Paramita", "Septelia Inawati Wanandi", "Novi Silvia Hardiany", "Purnamawati Huang", "Syarifah Dewi", "Reni Paramita" ], "abstract": "Background: Glioblastoma multiforme (GBM) is the most aggressive form of malignant glioma and is also known as grade IV astrocytoma. This might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal. Recently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth. Until now, the role of MSC secretomes in cancer stem cell pluripotency remains unclear. The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells. Methods: Umbilical cord-derived MSCs (UCSCs) were grown on serum-free alphaMEM for 24 hours to prepare the UCSC-CM. Human GBM T98G cells were treated with UCSC-CM for 24 hours. Following this treatment, expression of pluripotency markers SOX2, OCT4 and NANOG genes was analyzed using quantitative RT-PCR. Results: SOX2 and OCT mRNA expression was 4.7-fold (p=0.02) and 1.3-fold (p=0.03) higher in CM-treated cells compared to the control. However, there was no change in NANOG mRNA expression. This might be due to there being others factors regulating NANOG mRNA expression. Conclusions: UCSC-CM could affect the expression of SOX2 and OCT4 in human glioblastoma multiforme T98G cells. Further research is needed to elucidate the mechanism by which pluripotency markers are expressed when induced by the UCSC secretome.", "keywords": [ "conditioned medium", "mesenchymal stem cells", "glioblastoma multiforme", "pluripotency expression" ], "content": "Introduction\n\nGlioblastoma multiforme (GBM) is a primary brain tumor that arises from glial cells (glioma), and is the most aggressive form of malignant glioma. According to the WHO, GBM is also known as grade IV astrocytoma1. The life expectancy of patients with GBM is very low, usually less than 1 year despite available options such as surgery and chemoradiation. Failure of therapy might be due to the presence of cancer stem cells with high pluripotency and ability of self-renewal2. Cancer stem cells have an improved the ability to repair their own DNA3, and can be identified by the analysis of transcription factors found in embryonic stem cells such as OCT-4 (octamer-binding transcription factor 4), SOX2 [SRY (sex determining region Y)-box 2] and NANOG. Those transcription factors play an essential role in sustaining the pluripotency and self-renewal ability of embryonic stem cells4,5.\n\nRecently, it has been reported that tumor stroma cells, including mesencyhmal stem cells (MSCs), secrete factors that affect cancer cell growth. Previous studies have demonstrated that MSCs were recruited from bone marrow and that they home around the cancer cells to support tumor growth and metastasis6. Other studies have shown that MSCs can trigger cancer growth by inducing angiogenesis, suppressing the immune system, forming cancer-associated fibroblasts (CAF) that contributing to the tumor growth, epithelial mesenchymal transition (EMT) and metastasis7. The role of MSCs in cancer growth has been widely investigated. Nevertheless, the role of MSC secretomes in cancer stem cell pluripotency remains unclear. The aim of this study was to analyze the effect of MSC secretomes in conditioned medium (CM) on the expression of pluripotency markers of GBM cells.\n\n\nMethods\n\nThe Human Glioblastoma Multiforme T98G cell line was grown in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) on T-25 cm2 culture flasks (Corning). Medium was added with sodium bicarbonate, 10% fetal bovine serum (FBS, Biowest), 1% streptomycin - penicillin and 1% amphotericine at 37°C in a humidified atmosphere of 95% air and 5% CO2. The medium was changed 2 times in a week. T98G cells were sub-cultured after being 70–80% confluent8.\n\nUmbilical cord-derived MSCs (UCSCs) were kindly provided by Prof. Jeanne Adiwinata Pawitan (Cell Medical Technology Integrated Service Unit, Cipto Mangunkusumo Central Hospital, Jakarta)9. 125,000 UCSCs were cultured in Minimum Essential Medium alpha (αMEM, Gibco) supplemented with 10% FBS (Biowest)/Glutamax (Gibco), 1% streptomycin-penicillin and 1% amphotericin on T-25 cm2 culture flasks (Corning) at 37°C in a humidified atmosphere of 95% air and 5% CO2. After the cells were 70–80% confluent, medium was removed and cells were washed 3 times using 1x Phosphate Buffer Saline (PBS). Then, cells were grown on serum free- αMEM for 24 hours to prepare the UCSC-CM. After 24 hours, UCSC-CM were collected, centrifuged to remove cell debris and passed through 0.22 µm filter. Concentration of UCSC-CM was 50%, diluting UCSC-CM in freshly high glucose DMEM.\n\n400,000 T98G cells were seeded in triplicate on a 12-well-plate in high glucose DMEM/10% FBS/1% streptomycin-penicillin/1% amphotericin and allowed to adhere overnight. The following day, medium of T98G cells was replaced by 50% (v/v) UCSC-CM and incubated for 24 hours.\n\nSOX2, OCT4 and NANOG genes expression was detected using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). First, total RNA was extracted from CM-treated T98G cells using Tripure RNA Isolation kit (Roche). Total RNA was amplified using qPCR (PCRmax Eco48, United Kingdom) with KAPA SYBR FAST One step qRT-PCR (KAPA Biosystem) and primers for SOX2, OCT4, and NANOG (Table 1). Reaction protocol was as follows: cDNA synthesis for 10 minutes at 42°C, inactivation with iScript Reverse transcriptase for 5 minutes at 95°C, then 40 PCR cycles for 10 seconds at 95°C, followed by 30 seconds at 57°C for OCT4, 59°C for SOX2, 60°C for NANOG; then 30 seconds at 72°C. Following the PCR cycles, the protocol was subsequently continued with the melting curve analysis, i.e. 1 minute on 95°C; 1 minute on 55°C; 10 seconds on 55°C (80 cycles, increase 0.5°C every cycles).\n\nAll data were presented as means ± SD from triplicate experiments. Statistical analysis was performed using Student’s t-test using PASW 18 software, with p < 0.05 as a cut-off for determining a significant difference.\n\n\nResults\n\nCell morphology was observed under an inverted microscope (100X magnification) after 24 hours of the CM treatment. There was no difference in morphology between control and CM-treated T98G cells, as shown in Figure 1. Both control T98G cells and CM-treated T98G cells appear with fibroblast morphology, adhering to the plate. There was no change in cell shape and the cell membrane was still intact.\n\nAbout 4 × 105 cells were plated triplicate in a 12-well plate and grown in high glucose Dulbecco’s Modified Eagle’s Medium supplemented with 10% FBS/1% penicillin-streptomycin/1% amphotericin at 37°C, 5% CO2 for 1 day. Afterwards, the medium of treated cells was replaced with 50% (v/v) conditioned medium of umbilical cord stem cells, while medium of control cells was replaced with 50% (v/v) Minimum Essential Medium alpha. After 24-hour incubation, cell morphology was observed under inverted microscope (100X magnification). (A). Control cells; (B). Conditioned Medium-treated cells.\n\nPluripotency marker expression was analyzed by measuring SOX2, OCT4 and NANOG mRNA expression. Relative mRNA expression was calculated using the Livak method (2-ΔΔCT) with 18S rRNA as the reference gene. SOX2 mRNA expression was significantly higher in CM-treated T98G cells compared to control (Figure 2). SOX2 mRNA expression was up-regulated 4.7-fold in CM-treated T98G cells. Moreover, OCT4 mRNA expression was also significantly increased in CM-treated T98G cells compared to the control (Figure 3). OCT4 mRNA expression increased 1.2 fold in CM-treated T98G cells. Nevertheless, there was no significant change in NANOG mRNA expression (Figure 4).\n\n100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect SOX2 mRNA expression. The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene. All values are means ± SE, n = 9. Significant differences at (p<0.05). SOX2 expression was 4.7-fold (p=0.02) higher in the conditioned medium-treated T98G cells compared to the control.\n\n100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect OCT4 mRNA expression. The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene. All values are means ± SE, n = 9. Significant differences at (p<0.05). OCT4 expression was 1.3-fold (p=0.03) higher in the conditioned medium-treated cells compared to the control.\n\n100 ng of total RNA was amplified using quantitative reverse transcriptase polymerase chain reaction to detect NANOG mRNA expression. The expression was relatively calculated using Livak formula with 18S rRNA gene as a reference gene.All values are means ± SE, n = 9. Significant differences at *(p<0.05).There was no significant difference in NANOG expression between control and conditioned medium-treated T98G cells.\n\n\nDiscussion\n\nSimilar to normal stem cells, stem cell-like properties such as pluripotency in glioma CSCs are maintained by a core set of transcription factors, including SOX2, OCT4, and NANOG. Up-regulation of this set of genes is associated with poor outcome in terms of tumor malignancy, recurrence and metastasis11. Here, we demonstrated that the pluripotency markers SOX2 and OCT4 were up-regulated in UCSC-CM-treated cells. This indicates that UCSCs secrete certain factors that support the self-renewal capacity of GBM cells. Liu et al showed that bone marrow-derived MSCs produce a cytokine meshwork that stimulates CSCs12.\n\nThere is accumulating evidence suggesting that the regulation of stem cell-like properties requires a two-way interaction between CSCs and their microenvironment, particularly the MSCs. For instance, secretion of interleukin-1 (IL-1) in colon cancer cells could stimulate MSCs to produce prostaglandin E2 (PGE2). Then, PGE2 collaborated with IL-1 to produce other cytokines and chemokines such as IL-6, CXCL1 & CXCL8 by MSCs, leading to enhancement of the cancer stem cell population13. Wu et al proved that cytokines (IL-6, CXCL-8) were detected in conditioned media from MSCs. Those cytokines stimulated the expression of pluripotency markers (SOX2, OCT4, cMyc), as well as NF-κB &AMPK/mTOR signaling pathways in colon cancer cells14. In another study by Luo et al., CCL5 secreted by recruited BM-MSCs were found to induce prostate CSCs via androgen receptor signaling15. Investigating the secreted components of our UCSC-CM could allow us to determine more targeted CSC therapy in GBM.\n\nUnlike SOX2 and OCT4 expressions, NANOG mRNA expression in T98G cells was not affected by UCSC-CM in our study. This is might be due to the abundance of NANOG pseudogenes16,17 and also due to the way NANOG mRNA undergoes unique N6-methyladenosine (m6A) posttranscriptional modification as part of its regulation18. Furthermore, microRNA-134 has been reported to suppress proliferation and invasion of T98G cells by reducing NANOG expression19. Further studies are required to elucidate the involvement of UCSC secretomes in inducing the differential expression of pluripotency markers.\n\n\nConclusions\n\nThe conditioned medium of umbilical cord-derived mesenchymal stem cells could affect the expression of SOX2 and OCT4 as pluripotency markers in human glioblastoma multiforme T98G cells.\n\n\nData availability\n\nDataset 1: 18S rRNA Cq values. 18S rRNA Cq was used to calculate SOX2, OCT4 and NANOG mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d19129620\n\nDataset 2: SOX2 Cq values. SOX2 Cq was used to calculate SOX2 mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d19129721\n\nDataset 3: OCT4 Cq values. OCT4 Cq was used to calculate OCT4 mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d19129822\n\nDataset 4: NANOG Cq values. NANOG Cq was used to calculate NANOG mRNA expression using the Livak formula (K: control cells; CM: condition medium-treated cells). DOI, 10.5256/f1000research.13154.d19129923\n\nDataset 5: Raw unedited images for Figures 1A and 1B. DOI, 10.5256/f1000research.13154.d19130024", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by the Penelitian Unggulan Perguruan Tinggi (PUPT 2017) grant provided by the Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia (DRPM-UI).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nThe authors would like to express our gratitude to Direktorat Riset dan Pengabdian Masyarakat Universitas Indonesia (DRPM-UI) for the Penelitian Unggulan Perguruan Tinggi (PUPT 2017) grant. Moreover, thank you to PT. Ecosains Hayati, Indonesia for the assistance in providing real time PCR equipment.\n\n\nReferences\n\nLouis DN, Ohgaki H, Wiestler OD, et al.: The 2007 WHO classification of tumours of the central nervous system. Acta Neuropathol. 2007; 114(2): 97–109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolmberg J, He X, Peredino I, et al.: Activation of neural and pluripotent stem cell signatures correlates with increased malignancy in human glioma. Plos One. 2011; 6(3): e18454. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBao S, Wu Q, McLendon RE, et al.: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature Lett. 2006; 444(7120): 756–760. PubMed Abstract \| Publisher Full Text\n\nFong H, Hohenstein KA, Donovan PJ: Regulation of self-renewal and pluripotency by Sox2 in human embryonic stem cells. Stem Cells. 2008; 26(8): 1931–1938. PubMed Abstract \| Publisher Full Text\n\nLoh YH, Wu Q, Chew JL, et al.: The Oct4 and nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Gen. 2006; 38(4): 431–440. PubMed Abstract \| Publisher Full Text\n\nChaturvedi P, Gilkes DM, Wong CC, et al.: Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. J Clin Invest. 2013; 123(1): 189–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee JK: Mesenchymal stem cell: a hard nut to crack in cancer development. Enliven Archive. 2014; 1. Reference Source\n\nHardiany NS, Sadikin M, Siregar NC, et al.: The suppression of manganese superoxide dismutase decreased the survival of human glioblastoma multiforme T98G cells. Med J Indones. 2017; 26(1): 19–25. Publisher Full Text\n\nPawitan JA, Kispa T, Mediana D, et al.: Simple production method of umbilical cord derived mesenchymal stem cell using xeno-free materials for translational research. J Chem Pharm Res. 2015; 7(8): 652–656. Reference Source\n\nTachi K, Shiraishi A, Bando H, et al.: FOXA1 expression affects the proliferation activity of luminal breast cancer stem cell populations. Cancer Sci. 2016; 107(3): 281–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu A, Yu X, Liu S: Pluripotency transcription factors and cancer stem cells: small genes make a big difference. Chin J Cancer. 2013; 32(9): 483–487. PubMed Abstract \| Free Full Text\n\nLiu S, Ginestier C, Ou SJ, et al.: Breast cancer stem cells are regulated by mesenchymal stem cells through cytokine networks. Cancer Res. 2011; 71(2): 614–624. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi HJ, Reinhardt F, Herschman HR, et al.: Cancer-stimulated mesenchymal stem cells create a carcinoma stem cell niche via prostaglandin E2 signaling. Cancer Discov. 2012; 2(9): 840–855. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWu XB, Liu Y, Wang GH, et al.: Mesenchymal stem cells promote colorectal cancer progression through AMPK/mTOR-mediated NF-κB activation. Sci Rep. 2016; 6: 21420. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo J, Lee SO, Cui Y, et al.: Infiltrating bone marrow mesenchymal stem cells (BM-MSCs) increase prostate cancer cell invasion via altering the CCL5/HIF2α/androgen receptor signals. Oncotarget. 2015; 6(29): 27555–27565. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJeter CR, Liu B, Liu X, et al.: NANOG promotes cancer stem cell characteristics and prostate cancer resistance to androgen deprivation. Oncogene. 2011; 30(36): 3833–3845. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKawamura N, Nimura K, Nagano H, et al.: CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells. Oncotarget. 2015; 6(26): 22361–22374. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang C, Samanta D, Lu H, et al.: Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA. Proc Natl Acad Sci U S A. 2016; 113(14): E2047–E2056. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNiu CS, Yang Y, Cheng CD: MiR-134 regulates the proliferation and invasion of glioblastoma cells by reducing Nanog expression. Int J Oncol. 2013; 42(5): 1533–1540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHardiany NS, Huang P, Dewi S, et al.: Dataset 1 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source\n\nHardiany NS, Huang P, Dewi S, et al.: Dataset 2 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source\n\nHardiany NS, Huang P, Dewi S, et al.: Dataset 3 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source\n\nHardiany NS, Huang P, Dewi S, et al.: Dataset 4 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source\n\nHardiany NS, Huang P, Dewi S, et al.: Dataset 5 in: Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells. F1000Research. 2018. Data Source" }	[ { "id": "32142", "date": "26 Mar 2018", "name": "Jie Sun", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study mainly showed the effects of MSC conditioned medium in glioblastoma multiforme cells. Authors found that MSC-CM promote the expression of pluripotency markers in T98G cells, like SOX4 and OCT4, but not NANOG. The research has limited novelty and integrity. Main concerns are listed below:\nSerum free medium were used to acquire the MSC-CM for 24 hours in this study, authors should test the cell viability of MSC in this state for a better assess the quality of CM.\n\nAll the gene markers were tested in mRNA level in the manuscript, protein levels should be added.\n\nOnly a small number of cells were regarded as cancer stem cells, especially in commercial cell lines. MSC-CM increases the original stem cells to express pluripotency markers or it could induce non-stem cells to express these markers still unclear. What is more, whether CM would affects cell functions after the increase of these markers still unclear. All these date are needed to be completed or discussed in the manuscript.\n\nAs authors mentioned in the discussion, various researches has been tested to clarify the interactions between MSC and cancer cells. Based on the existed results in this paper, limited novelty also decrease the quality of the manuscript.\n\nThe background of the pictures in Figure 1 should be changed and the scale bar should be added.\n\nThe statistical analysis should not be used on its own, as the study is not good and qualified enough/\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "33378", "date": "10 May 2018", "name": "Isabele C. Iser", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the study titled “Analysis of pluripotency marker expression in human glioblastoma multiforme cells treated with conditioned medium of umbilical cord-derived mesenchymal stem cells”, the authors investigated the effects of UCSC on the expression of pluripotency factors in GBM cells. The authors showed an increased expression of SOX4 and OCT4 in GBM cells treated with CM when compared to untreated cells. The work presents numerous limitations, and does not have scientific relevance that justify its indexing. For example:\n\nThe authors evaluated the gene expression only at the RNA level, however, the ideal would be to evaluate also at the protein level in order to obtain more conclusive results. In addition, the authors evaluated a limited number of genes related to CSCs. I suggest evaluating a greater number of genes to confirm the results presented here. The authors also should access other parameters in the cells besides gene expression, such as resistance to therapy, proliferation and viability. It is also very important analyze which factors in the CM could be responsible for the effect presented in this work. I strongly suggest that the authors use a positive control for qPCR reactions, such as GBM cells treated with CSC inducers. In the discussion the authors say that “the up-regulation of this set of genes is associated with poor outcome in terms of tumor malignancy”, so in my opinion it would be interesting to perform an in vivo model to testing the effects of these treated cells in terms of tumor formation and malignancy.\n\nIn view of these considerations, I do not consider the work apt to be published in F1000 Research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/7-106
https://f1000research.com/articles/6-1461/v1	14 Aug 17	{ "type": "Research Article", "title": "BRCA1 novel mutation V1736D and in silico analysis of SNP Q356R in Sudanese patients with breast cancer", "authors": [ "Mohamed Elmogtba Mouaweia Mohamed Aabdein", "Alsmawal Awad Mohammed Elimam", "Hisham N. Altayb", "Mohamed El-Fatih Mohy Eldeen", "Mosab Mohamed Gasemelseed", "Afra AbdElhamid FadlAlla", "Marwa Mohamed Osman", "Soada Ahmed Osman", "Hajir Ali Saeed", "Mona ShamsAldeen Ali", "Tomador Siddig", "Reem Abdelrahman Osman", "Rehab Ahmed Elhadi", "Muzamil Mahdi Abdel Hamid", "Mohamed Ahmed Salih", "Hisham N. Altayb", "Mohamed El-Fatih Mohy Eldeen", "Mosab Mohamed Gasemelseed", "Afra AbdElhamid FadlAlla", "Marwa Mohamed Osman", "Soada Ahmed Osman", "Hajir Ali Saeed", "Mona ShamsAldeen Ali", "Tomador Siddig", "Reem Abdelrahman Osman", "Rehab Ahmed Elhadi", "Muzamil Mahdi Abdel Hamid", "Mohamed Ahmed Salih" ], "abstract": "Background: Breast cancer (BC) remains one of the leading causes of death in women worldwide. The BRCA1 deleterious mutation has a significant role in developing BC, and the risk has been estimated to be 46–87%. Many studies emphasize the need for mining BRCA1 gene mutations that might have a role in BC pathogenesis and could affect early disease onset. This study was conducted to screen for possible pathogenic single nucleotide polymorphisms (SNPs) in BRCA1, targeting three regions: two in exon 11 and the third in exon 20. Methods: 45 blood samples were collected from patients diagnosed with BC. DNA was extracted and selected regions were amplified by PCR using three sets of primers - two within exon 11 and one within exon 20 of BRCA1. Subsets of 10 samples were selected for each primer set (30 PCR products) and sequenced. Sequences were analyzed using various bioinformatics tools. Results: Two missense mutations were found, Q356R (rs1799950) in one patient (27 years old) and a novel SNP, V1736D, in three premenopausal patients (≤45 years), which were located within exons 11 and 20, respectively. Both detected variants were heterozygous, a status found in all patients detected with such monoallelic variation. Both missense variants underwent in silico analysis. The well-known mutation, rs1799950, was predicted to alter the protein activity, conferred by a mutant residue (R-Arg), owing to the position with a bigger size and positive charge. The novel SNP, V1736D, was predicted to play a role in the pathogenesis of BC. Conclusion: Both variants require further investigation, firstly to assess their contribution to BC and secondly to determine their potential diagnostic value when assessed in a larger population.", "keywords": [ "breast cancer", "BRCA1 gene", "SNP", "novel mutation", "missense variants", "heterozygous", "Sudanese patients" ], "content": "Introduction\n\nBreast cancer (BC) is a very serious issue worldwide, and is one of the leading causes of death in women today. In the US, it was estimated that there were approximately 232,670 new cases of BC and 40,000 BC deaths in 2014, and the number increased with 13,990 new cases and 450 deaths in 20161,2. In Africa, in 2012, the rate was about 94,000 women with BC, which resulted in 48,000 deaths1,2, and studies in Africa have described a poor outcome with a late diagnosis, due to the aggressiveness of the disease and the absence of screening programs3–6.\n\nIn Sudan, BC occurs at the highest frequency among women compared to other types of cancer7–12. In a hospital-based statistical report in Sudan12, BC was found to be the most commonly diagnosed malignant tumor and was characterized by early onset and bad prognosis. The report showed invasive ductal carcinoma to be the predominant type (82%), and 74% of patients were <50 years old with an advanced disease stage, indicating that most cases remain undiagnosed for long periods7,8,12–14. BRCA1 (OMIM_113705) was mapped in 1994 and subsequently cloned. It is located on chromosome 17 region 2, band 1 (17q21), which is responsible for encoding 1863 amino acids15. Since 1995, the BRCA1 tumor suppressor protein has been found to arrest cell proliferation, play an important role in the repairing process of DNA damage, and was suggested to have a role in cell cycle regulation through interacting directly or indirectly with other regulatory molecules16–21. BRCA1 when it is altered becomes deficient, and such loss of function mutates the protein, which not only perturbs chromosomal integrity and genome stability, but increases the mutation rate of other genes22–25. Therefore, it has been proposed that BRCA1 doesn’t directly initiate cancer formation, but enhances the process by making the affected cells highly susceptible to malignant transformation26,27. Germline mutations in BRCA1 are responsible for a large proportion of inherited predispositions for BC, and individuals that carry an inherited mutation in the BRCA1 gene have a significant risk (46–87%) for developing BC by 70 years of age28–33. Regionally among Africa and locally in Sudan, BRCA1-associated BC has been identified among premenopausal women5,8,34. This indicates the highly susceptible nature of such a mutation to enhance cancer development earlier during fertile reproductive women. African scientific literature has greatly studied the risk of BC resulting from reproductive factors (such as early menarche, late menopause, and sex hormones), but has not explored much into the genetic predisposition of the disease5,35–37.\n\nAbout 13108 SNPs have been reported within human BRCA1, of them 1608 were reported as missense variants and about 151 have been identified to be pathogenic, according to the SNP database at NCBI (http://www.ncbi.nlm.nih.gov/snp). Most of these mutations resulted from small insertions/deletions, leading to frameshift, stop codon or nonsynonymous missense substitution, deletion, duplication and disruption of splice sites, resulting in a nonfunctional protein38. The SNP variant rs1799950 was observed to have a negative association with BC, by favoring more frequently the control groups than BC patients39,40. This SNP was tested among familial BRCA1 carriers for BC risk association, and a significant association was found within affected families41. When the SNP was haplotype-homozygous within affected families, the risk was increased, as in the case of sporadic risk association study42. The hetero-homozygosity nature of this mutation has been noticed within different studies, and some studies found the heterozygous variant more frequently within controls, hence it was adversely associated with BC, while in another study, the homozygous variant was found more frequently within BC patients39,42. In addition, heterozygosity was functionally assessed among monoallelic BRCA1 mutation carriers of rs1799950, and the results showed that such an alteration could permit variation in protein expression and activity in a haploinsufficient way, which could alter the cell’s normal behavior and result in tumor transformation by enhancing tumorigenesis. Such variation conferred by one mutated copy suggests that the wild-type copy alone is not capable of compensating the loss of the other wild allele43,44. Turkovic et al. found that such a haplotype association was noticed more frequently within deleterious mutation carriers; however, this was observed in a small sample size45. In addition, some studies have found rs1799950 to be associated with early-onset prostate cancer46–48.\n\nTwo genetic studies have been conducted in Sudan concerning BRCA1. One was a survey of 2370 students at a girl’s secondary school in Northern Sudan-Marawi, in which the study divided 67 students into two groups (47 students with a family history of BC and 20 with unaffected families) to analyze BRCA1 and BRCA2 mutations. In the first group, which was 2.37% of responders, the frequency of mutations was higher for BRCA1, and most mutations were within exon 11. The study continued to recommend further assessments of this region in subsequent local projects, and this formed the basis of our primer (1 and 2) selection within the present study9. The other study, from central Sudan, investigated 34 early onset premenopausal women patients diagnosed with BC (<40 years) and one male patient. The study identified 60 mutations in these patients, five of which were deleterious, affecting the outcome protein8.\n\nFrom the same region within central Sudan, early onset BC premenopausal women have also been investigated for BRCA1 point mutations. The findings revealed the presence of one deleterious variant, 24 neutral variants and eight variants of unknown significance, within which two novel variants were discovered34.\n\nSince there have been scarce genetic studies conducted highlighting genetic characteristic and familial risk status of BC patients in Africa (37) our aim was to screen for the type and spectrum of germline mutations in BRCA1 by focusing on three regions within the gene, two within exon 11 and one within exon 20, using sequencing, and to further assess the detected variants using in silico analysis tools. These regions and their selections were based on the quality of available primers (e.g. best GC content, adequate length, according to previous literature49), previous local research findings revealing frequent mutations within exon 118,9, and the cost.\n\n\nMethods\n\nThis study was carried out in March 2015 at the Radiation and Isotope Center in Khartoum. 2–3 mls of blood were collected randomly from 45 patients diagnosed with BC who attended the center for treatment and follow-up (no other inclusion/exclusion criteria were relevant), using sterile EDTA-K3 vacutainer and kept at -20°C. All the patients were aged between 27 and 80 years old, with a mean of 45.9 years. Early onset cases were more frequent than late onset: 25 (55.6%) cases with early onset, with a mean of 36.6 years; and 20 (44.4%) cases of late onset with a mean of 57.4 years. Multiparity was high in 30/45 (66.6%). Six cases reported a family history of BC (13.3%), abortion was detected in 10 cases (22.2%). Of the presented histotypes that were available to us (16 cases unknown), ductal type tumor was more frequent and shown in 22 cases (48.8%), then lobular in five cases and mucinous in two cases. In addition, five cases showed distal cancer metastasis; lung and bone were frequent secondary sites. Right side tumor originated in 20 cases (44.4%), while the left side tumor was found in 15 cases (33.3%), and four cases presented with bilateral BC (6 cases unknown) (see Table 1).\n\n*Comprising both Khartoum 16 cases and AlGezirah 5 cases.\n\nThis study was conducted under the guidelines and approval of the Research Ethics Committee of Sudan Ministry of Health – Khartoum state. All participants provided oral informed consent to participate in the study. Oral informed consent was obtained as opposed to written consent, due to the literacy levels of the patients and the time limited interaction between researchers and patients at the hospital.\n\nDNA was extracted using the salting-out method50 for 45 patients samples. In addition, proteinase K was used to enhance WBC membrane breakdown at 56°C for 1 hour. For PCR, three previously published pairs of primers49 were used to amplify three regions within the BRCA1 gene. All the three primers were selected for their quality performance, optimal size and GC content, after being assessed with Oligoanalyzer tool 3.1 (https://www.idtdna.com/calc/analyzer). These primers were synthesized by Macrogen Incorporation (Seoul, South Korea; Table 2). Annealing temperatures were adjusted on several runs (Table 2). Maxime PCR PreMix Kit i-Taq 20 μl (INTRON Biotechnology, South Korea) was used for PCR - 15 ul distilled water, 3 ul sample DNA (30 ng/ul; as checked by NanoDrop 1000) and 1 ul of the final concentration of each primer (10 pmol/µl forward and reverse). PCR mixture was subjected to initial denaturation step at 96°C for 5 minutes; followed by 35 cycles of denaturation at 96°C for 30 seconds, primer annealing at 50 or 55°C depending on the set used, for 30 seconds; followed by a step of elongation at 72°C for 60 seconds; the final elongation was at 72°C for 10 minutes49. After PCR amplification, the PCR products (442, 271 and 401bp) were checked by 2% gel electrophoresis at 100 V for 30–45 min (Figure 1).\n\nLeft-side, PCR amplicons of primer 2, size 271 bp; right-side, PCR amplicons of primer 3, size 401 bp; L, ladder of 100bp each.\n\nF: forward; R: reverse; bp: base pair; CDS: coding sequence; V1: variant one.\n\nThe product size of the first and last primers were checked and assessed using Serial Cloner version 2.6.1 (http://serialbasics.free.fr/Serial_Cloner.html) on the known nucleotide database accession gene for BRCA1 (NG_005905) with the whole sequence size of 81189bp, both forward and reverse of each one have been found to determine regions that cover coding and non-coding sequences.\n\nThe NCBI RefSeqGene NG_005905, which represents the whole BRCA1 gene, and the transcript variant 1 NM_007294 mRNA, which comprises mainly the coding sequences of the gene. The first sequence was used to check that all three primers amplicons within the BRCA1 gene region, while the second sequence was used for assessing all the three primers amplicons within the BRCA1 coding sequence. The gene sequence of BRCA1 has marked all the primers set amplicons to be within the gene region sequence. The transcript variant 1 (NM_007294) mRNA has marked (only 367 and 86bp) nucleotide sequences within primers sets 1 and 3 amplicons, respectively, to be within coding sequences, and the whole set of primer 2 amplicon (271bp) was within the coding sequence of the BRCA1 gene (Table 2).\n\nThe PCR products of the 10 best bands yielded from the patient samples for each primer set, a total of 30 resulted amplicons, were sent for Sanger dideoxy sequencing. Partial standard sequencing for the three regions within the gene, including both forward and reverse nucleotide sequencing, was performed by Macrogen Company (Seoul, South Korea), using the same pairs of primers.\n\nSequence analysis. The sequence results for the 30 sequence chromatogram files were viewed by FinchTV program version 1.4.051, which was used to check both nucleotide sequences of the patients forward and reverse sequences to be free of errors. Any errors were excluded during processing. The Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to assess nucleotide and protein sequence similarities52. In ExPASy translate tool - SIB Bioinformatics Resource Portal, the gene sequences were translated into amino acid sequences53. For primers 2 and 3, the BRCA1 nucleotide sequences from the patients, with their translated proteins, underwent multiple sequence alignment using BioEdit software version 7.0.9.154. Multiple sequence alignment included the reference sequence with the highest similarity, as obtained by BLAST (RefSeq transcript mRNA - NM_007294 transcript variant 1), two additional nucleotide sequences (NM_007297.3, transcript variant 3 mRNA and JN686490.1; Figure 2A), and the gene sequence NG_005905, which is mainly the sequence between positions 68120-68810 (Figure 2B).\n\n(A) Heterozygous substitution from Glutamine (Q) to Arginine (R) at position 356 in patient 23, due to missense substitution mutation from Adenine (A) to Guanine (G) c.1299A>G, coding sequence. (B) Heterozygous substitution from Valine (V) to Aspartic acid (D) at position 1736 in patients 2, 22 and 26 due to missense substitution mutation from thymine (T) to adenine (A) c.5439 T>A coding sequence. Using the transcript variant 1 NM_007294 mRNA, which represents the complete BRCA1 coding sequence used to align all patient sequences under screening, and the corresponding amino acid sequences NP_009225 to align all patient translated amino acid sequences.\n\nSNP information. SNP information [SNP ID, MIM: 113705, RefSeq Gene accession No.: NG_005905 on chromosome 17, mRNA accession NO.: NM_007294 transcript variant 1 with 7224 bp and Protein accession numbers : (NP_009225) protein isoform 1 with 1863 a.a. and P38398 UniProt entries] concerning the human BRCA1 gene, which was used in our computational analysis, was retrieved from the NCBI database of SNPs: dbSNP (https://www.ncbi.nlm.nih.gov/snp).\n\nSNP prediction. SNPs were analyzed using five prediction online tools: SIFT (http://sift.bii.a-star.edu.sg/)55, Polyphen-2 (http://genetics.bwh.harvard.edu/pph2/)56, I-MutantDDG-Seq Suite (http://gpcr2.biocomp.unibo.it/cgi/predictors/I-Mutant3.0/I-Mutant3.0.cgi) and PhD-SNP (http://snps.biofold.org/phd-snp/phd-snp.html)57. The tertiary model of protein and mutation analysis was done online using Hope software (http://www.cmbi.ru.nl/hope/input)58. MutationTaster2 (http://www.mutationtaster.org/) was used to assess the protein features of the resulting variants, with comprehensive (input/output) criteria, which predicts potential disease-causing mutations59.\n\n\nResults\n\nTwo missense variants were detected within the study, one patient with Q356R and three patients with V1736D. Both variants were heterozygous (Figure 2) and were detected within premenopausal patients, with a mean age of 37 years. Three patients were multiparous; the one case of (Q356R) and two cases of (V1736D) were multiparous (mean parity, 2.8). There was no family history of BC in patients with the two variants (Table 3).\n\nThe patients’ sequences, and the additional nucleotide sequences with the gene sequence, were aligned against the reference standard sequence in the RefSeqGene and nucleotide databases (accession, NM_007294), which is BRCA1 gene transcript variant 1, and was introduced to replace the previously existing sequence (gi: 63252871) in May 200960. Two substitutions-bearing monoallelic alterations were found, the first one at position 1299 (A/G) located in exon 11 (Figure 2), and the second one at position 5439 (T/A) in exon 20 (Figure 2). Primer 1 has been excluded from the study because of the errors that have been noticed within all patient sequence data chromatogram results.\n\nAfter translation to amino acid sequences, the samples were aligned against BRCA1 protein isoform 1 (accession, NP_009225). Q356R was found to meet its corresponding nucleotide change and position c.1299A>G in which Glutamine (Gln) replaced by Arginine (Arg) in patient 23, and V1736D was found to meet its corresponding nucleotide change and position c.5439T>A, in which Valine (Val) was replaced by Aspartic acid (Asp) in patients 2, 22 and 26 (Figure 2). These variants (Q356R, V1736D) were then predicted with SIFT, Polyphen-2, I-Mutant-3, PhD-SNP and MutationTaster2 software to obtain their pathological effects, and results are provided in Table 4. Amino acid properties for the wild Val and the mutant Asp residues and the 3D structure of the variant V1736D were obtained using Project Hope software (Figure 3A).\n\nSNP: single nucleotide polymorphism; RI: reliability; DDG: ΔΔG; SVM: support vector; SVM2 value: DDG < 0: decrease stability, DDG >0 increase stability machine; DDG value: DG (New Protein)-D (Wild Type) in Kcal/mole.\n\n(A) This illustration shows the BRCA1 protein in grey colour on the (left) with the magenta coloured side chain of the mutated residue, and on the (right) the higher magnification of the area of mutation displays the side chains of both amino acid residues more clearly, the green highlights the wild type Valine, and the red highlights the mutant type Aspartic acid with their schematic structures, as both contain the same backbone (red) but they differ in their side chains (black). (B) The illustration shows the schematic structural units of the neutral wild type Glutamine (left) and the positive mutant Arginine (right). Both amino acids contain the same backbone (Red) but they differ in their side chains (Black).\n\n\nDiscussion\n\nIn this study, we found two mutations-bearing monoallelic features. One missense mutation rs1799950 in exon 11 BRCA1 gene in patient 23: position number 1299 (A/G) as (CAGA) to (CGGA), using transcript variant 1 (NM_007294), which led to change in the coding amino acid (Glutamine-Gln-Q) to (Arginine-Arg-R) at position 356 using protein isoform 1 (NP_009225.1) (Figure 2A).\n\nThe other was a novel SNP, situated at exon 20, with higher frequency in patients 2, 22 and 26: position 5439 (T/A) as (GTCA) to (GACA), which led to change in the coding amino acid (Valine-Val-V) to (Aspartic acid-Asp-D) at position 1736 (Figure 2B). Both SNPs (Q356R) and (V1736D) were found to affect the resulting translated protein. According to Project Hope software, starting with the novel (V1736D) variant, we found that the mutant residue Aspartic acid (D) of the outcome BRCA1 mutated protein is located near a highly conserved position. The mutant residue is bigger and less hydrophobic than the wild-type residue. The wild-type residue was neutral, the mutant residue is negatively charged. The mutated residue is located in a domain that is important for binding of other molecules, as it is in contact with residues in another domain. It is possible that the mutation can disturb these contacts. It may also disturb the interaction between these two domains, which would affect the function of the protein; therefore, it might disturb signal transfer from binding domain to the active domain. This novel mutation was detected in patients from different tribes from Sudan, who were all ≤45 years old.\n\nIn the case of the Q356R mutation, the mutant residue of Arg is positively charged compared to the neutral wild type Gln residue. This could lead to the repulsion of ligands or other similar charged residues. The size of the end variant is bigger than the wild-type residue and this might lead to bumps, as reported by Project Hope software (Figure 3B). In addition, the mutated residue is located on a domain responsible for protein activity; hence the activity could be altered by its physical variation conferred by its new charge and size. The Arg 356 variant does, however, generate a run of three positively charged residues (Lys Arg Lys), and as a result it could alter the properties of the protein, which is composed of 16% negatively charged residues overall61. In the current study, we found the same result when it was described as a disease-related mutation of altered protein stability (Table 4).\n\nAccording to MutationTaster2, the novel variant (V1736D) was predicted to be ‘disease-causing’, and the software assessed and calculated the probability of such variation on the resulting protein, and showed that the new features would be disease-causing and displayed it with a higher score. By constrast, the software predicted that in the case of Q356R, the resulting Arg was a polymorphism of little harm, and reported it with a lower score compared to the protein feature of the other variant, and reports from the other softwares used for assessment (Table 4). Q356R has not yet been classified in terms of clinical significance in NCBI dbSNP.\n\nTwo novel variants were identified to be deleterious within the BRCA1 gene among premenopausal women patients in two local studies8,34. One variant was a truncated stop codon and the other was predicted computationally: c.3999delT, stop codon 1335, and c.5090G>A, p.Cys1697Tyr, respectively. In addition to these variants, a deleterious mutation, c.4986+6T>C, located in intron-exon boundary was found in the youngest patient of 25 years in one of the studies34. These findings, compared to our present study finding (V1736D), showed that early onset BC is associated with a deleterious nature of the identified variants. In addition, our study included pre and postmenopausal patients, all detected variants were mainly confined to premenopausal cases. Therefore, genetic studies are highly recommended to highlight the genetically susceptibile nature of patients diagnosed with early onset disease, who may harbor the deleterious variants that could have a significant role in developing BC. Two patients within the present study were screened previously in a local study targeting pathological SNPs within BRCA2 gene selected regions, which identified a stop codon (L1053X) (Elimam, et al.; unpublished studya). One of these patients was the youngest patient identified with Q356R, and the other presented with the bilateral disease and identified with V1736D. Both patients were reported to have stop codons at position L1053X with nucleotide and protein sequences identifiers of KT901810 and ALQ44030, and KT901807 and ALQ44027, respectively (Elimam, et al.; unpublished studya).\n\nThe SNP found in the present study, c.1299A>G, matched a previously reported SNP, in the same altered nucleotide position 1299A>G, and the same altered outcome protein position, Q356R41,62. The same protein position (Q356R) with the same altered nucleotide (A/G) has also been described previously, but with a different nucleotide position c.1186 A>G15,63,64. Some studies found that this SNP is associated with patients under 40 years old,64,65, which agreed with our study - the youngest premenopausal patient (27 years old) was detected with this variant. Although this variant’s effect remains uncertain, a previous finding to our study has described the same mutation, Q356R in Moroccan BC patients, which was the first study that described this mutation in a North African population64. They found that most previous studies that described this mutation were within western European populations66 and there were no studies found in a North African population to confirm this, thus more research is needed to investigate this. Our study is closer to the Moroccan study regarding the early onset characteristic this variant has, revlead by the similarity in the background history of the patient with a Q356R polymorphism in our study and those detected in the Moroccan study, who also did not have a family history of BC64. This variant has been reported to be independently minor or leads to a very slightly increased BC risk, but a risk that is cumulatively significant67. In another study, this mutation was found in patients with a family history of ovarian cancer, suggesting that this variant may increase ovarian cancer risk63,68. Both detected variants in the present study were identified to have a pathological effect, with the exception of the results from MutationTaster2 in the case of Q356R. Therefore, the Q356R mutation may have a pathological effect, and the novel SNP might play a role in the pathogenesis of the disease.\n\nIn order to only have clear DNA sequence results for comparison with NCBI references, primer 1 results were excluded due to sequencing errors (see Supplementary File 1). In addition, three patient sequences of primer 2 set and one patient of primer 3 set have been excluded for the same sequence errors.\n\nThe limitation of this study was the small sample size and the functional assessment facilities available to assess the protein of monoallelic alteration for their pathological contribution to the disease. Financial constraints also limited the study. Therefore, we recommend further studies in a larger number of Sudanese patients to further explore these findings.\n\n\nConclusions\n\nIn the present study, Sudanese BC patients were investigated for BRCA1 mutations. Two different mutations were found in young patients of ≤45 years old, with no family history of BC. To conclude, the study has highlighted a need for further research of these mutations amongst a larger population (including patients and controls), so as to investigate the variants’ distribution through the population and their potential diagnostic value. This will aid the understanding of a variant’s frequency and clinical significance. In addition, both variants identified require in vitro functional and protein level assessment.\n\n\nData availability\n\nThe BRCA1 sequence data of the novel variant (V1736D) from this study has been submitted to NCBI GenBank under the accession numbers and protein identifiers found in Table S1.\n\nDataset 1: BRCA1 sequence result in a zipped file. These sequencing results as received from Macrogen Company (Seoul, South Korea) comprise all the breast cancer patients in this study using the three sets of primers (1, 2 and 3).Each patient has the sequencing data in different file formats (a sequencing data file that needs to be viewed by a sequencing viewer software, i.e FinchTV; a PDF; and a FASTA format text document). doi, 10.5256/f1000research.11395.d17244569\n\nDataset 2: Patient demographics (non-identifying) according to primer. Patient demographics, clinical data, and histological parameters with highlighted detected missense variants (primers 2 and 3) of each 10 selected subset patients. doi, 10.5256/f1000research.11395.d17244670\n\n\nFootnotes\n\naAuthors involved in the unpublished study: Alsmawal A. Elimam, Mohamed Elmogtba Mouaweia Mohamed Aabdein, Mohamed El-Fatih Moly Eldeen, Hisham N. Altayb, Mohamed Adel Taha, Mohammed N. Nimir, Mohamed D. Dafaalla, Musaab M. alfaki, Mohamed A. Abdelrahim, Abdelmohaymin A. Abdalla, Musab I. Mohammed, Mona Ellaithi, Muzamil Mahdi Abdel Hamid, Mohamed Ahmed Salih Hassan.", "appendix": "Author contributions\n\n\n\nMEMMA, AAME, MAS conceived the study. ME-FME, HNA, MMAH, MAS designed the experiment. MEMMA, AAME, sample collection and lab preparation. ME-FME, MMAH designed the wet lab practice. MEMMA, AAME, ME-FME practiced the wet lab methodology. MAS, HNA, AAME, MMG, AAF, MMO, SAO, HAS, MSA, TS, RAO, RAE bioinformatics assessment. AAME, MMG, AAF, MMO, SAO, MSA, TS prepared the manuscript draft. AAME, MMG, MSA, MMAH, MAS revised and edited the manuscript draft. MMAH revised the final edited manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors extend their heartfelt thanks to Prof. Mohamed Ahmed Salih for his priceless help and palatable advice, Al-Neelain University-Khartoum state, Radiation and Isotope center- Khartoum state and Africa city of technology-Khartoum state members for their help. And we would also love to thank Ms. Catherine Russel and Mr. Omar Awad Elimam for their kind support in language corrections.\n\n\nSupplementary material\n\nSupplementary File 1: Evidence for sequencing errors of primer 1. The file contains FinchTV snapshots, and the uncropped western blot for primer 1.\n\nClick here to access the data.\n\nTable S1: NCBI GenBank accession numbers for the novel BRCA1 sequence data from this study.\n\nClick here to access the data.\n\n\nReferences\n\nSiegel R, Ma J, Zou Z, et al.: Cancer statistics, 2014. CA Cancer J Clin. 2014; 64(1): 9–29. PubMed Abstract \| Publisher Full Text\n\nSiegel RL, Miller KD, Jemal A: Cancer statistics, 2016. CA Cancer J Clin. 2016; 66(1): 7–30. PubMed Abstract \| Publisher Full Text\n\nFregene A, Newman LA: Breast cancer in sub-Saharan Africa: how does it relate to breast cancer in African-American women? Cancer. 2005; 103(8): 1540–1550. PubMed Abstract \| Publisher Full Text\n\nAdesunkanmi AR, Lawal OO, Adelusola KA, et al.: The severity, outcome and challenges of breast cancer in Nigeria. Breast. 2006; 15(3): 399–409. 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Data Source" }	[ { "id": "24993", "date": "04 Sep 2017", "name": "Habiba Chaabouni-Bouhamed", "expertise": [ "Reviewer Expertise Genetics", "molecular genetics", "cytogenetics", "genetic counseling" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report the molecular analysis of 2 exons (11 & 20) of BRCA1 in a Sudanese population. The paper is well written.\nIt gives a truncated information about BRCA1 mutations in a population but the work is correctly done; it has to be completed by analysing the whole gene; using HRM method could help them to reduce cost analysis. One remark about reference 1& 2, I didn't find information about African statistics which are available in references 4 & 5.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "25416", "date": "14 Sep 2017", "name": "Mahmoud Balbaa", "expertise": [ "Reviewer Expertise Enzymes and cell signalling", "Protein expression", "gene expression." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBRCA1 was highly investigated around the whole world. The authors just applied the research of BRCA1 on Sudanese patients. Generally, the authors have obtained good results, but Figure 1 that shows the PCR amplification of the BRCA1 gene has a bad quality and should be changed to show clear band with a bp scale. The band of housekeeping gene e.g beta actin should be shown in the figure.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-1461
https://f1000research.com/articles/6-2020/v1	17 Nov 17	{ "type": "Opinion Article", "title": "Genetics in the 21st Century: Implications for patients, consumers and citizens", "authors": [ "Jonathan Roberts", "Anna Middleton", "Anna Middleton" ], "abstract": "The first human genome project, completed in 2003, uncovered the genetic building blocks of humankind. Painstakingly cataloguing the basic constituents of our DNA (‘genome sequencing’) took ten years, over three billion dollars and was a multinational collaboration. Since then, our ability to sequence genomes has been finessed so much that by 2017 it is possible to explore the 20,000 or so human genes for under £1000, in a matter of days. Such testing offers clues to our past, present and future health, as well as information about how we respond to medications so that truly ‘personalised medicine’ is now a reality.\n\nThe impact of such a ‘genomic era’ is likely to have some level of impact on all of us, even if we are not directly using healthcare services ourselves. We explore how advancements in genetics are likely to be experienced by people, as patients, consumers and citizens; and urge policy makers to take stock of the pervasive nature of the technology as well as the human response to it.", "keywords": [ "genomics", "patient", "citizen", "consumer" ], "content": "Introduction\n\nGenomic technology is now being utilised in more settings across society than ever before, including medicine, population health screening, recreational consumerism (ancestry testing, nutritional testing), through to policing and crime prevention. Signifying the importance placed on genomics, the most recent annual report of the Chief Medical Officer of the United Kingdom (2017) was entitled “Generation Genome” and stated:\n\n\"Genomics is not tomorrow. It’s here today. I believe genomic services should be available to more patients, whilst being a cost-effective service in the NHS. This is exciting science with the potential for fantastic improvements in prevention, health protection and patient outcomes. Now we need to welcome the genomic era and deliver the genomic dream!\"1\n\nAs members of the public we are all likely to all be exposed to some level of genomics within our lifetime – whether it be directly via a medical test, indirectly through marketing for recreational testing or distantly through being biologically related to someone whose DNA is contained within a national database. Given that genomic information links us to our relatives, the decisions that we make about it (whether to donate it for research, whether to be tested, whether to withhold it) will all have an impact on those we are related to and the knowledge that they too can gain. It is this fact that makes genetic information quite different from other sorts of medical information. Thus, we all have a stake in policy that guides the utilisation of genomic data. Within this opinion article, we reflect upon some of the ways that genomic data may touch us as patients, consumers and citizens.\n\nThe UK has been an early implementer of genomics in a healthcare setting. In 2014, the Deciphering Developmental Disorders Study reported the first results from the sequencing of 12,000 children with developmental disorders2. At the same time, work began on a project to sequence 100,000 genomes of 70,000 NHS patients3. Outside of the UK, sequencing in healthcare settings is also expanding. For example, the National Institutes of Health Precision Medicine Initiative in the US offers 1 million people some level of genomic sequencing4 and the Australian Genomics Health Alliance is currently creating the infrastructure to integrate genomic medicine into healthcare nationally. Possibly the most striking example comes form Qatar where the government has plans to offer genome sequencing to their entire population (www.qatargenome.org.qa/). Thus we can see that genomic technology is now being utilised globally in a wide range of contexts to provide diagnostic, prognostic and treatment information for patients using healthcare services in a way that is predicted to transform healthcare1.\n\nGenetic testing is currently used in a wide range of healthcare settings, including the diagnosis of rare disease in paediatrics, prenatal care, ophthalmology, dermatology, ENT, etc. Such testing will continue to be used in these settings. However, as genetic testing (single genes) shifts to genomic testing (many genes) and the ability to test becomes faster and cheaper, a key change that will occur is a dramatic increase in the amount of information generated. As an example, as recently as 5 years ago, a breast cancer patient who also has family history of young onset breast cancer would likely be tested in a healthcare setting for two genes (BRCA1 and BRCA2). Mutations in these genes are known to lead an increased risk for certain types of cancer, most notably breast and ovarian cancer. Whereas now, such a patient may have a ‘panel test’ where over 20 genes associated with breast cancer are explored5. Each of these genes will have different links to cancer, thus increasing the complexity of the results that emerge. In addition to using genomic technology to understand the genetic basis of an existing condition such as breast cancer, it can also be used to uncover a new diagnosis for a previously undescribed rare condition6. Again, here the key change is the volume of information that will be generated. Instead of testing just a few genes, a child with an unknown developmental disorder may now have 20,000+ genes sequenced and then filtered bioinformatically to explore all of the genes linked to intellectual disability, autism and developmental conditions. This may reveal new diagnostic or prognostic information. Whilst it is unrealistic to suggest that all 20,000 genes will be analysed and reported as a whole7, the resource is at least available to be interrogated as and when required. Genomic medicine is now available across whole healthcare systems, it has been truly ‘mainstreamed’6,8. Thus, patients have more exposure than ever before to the volume and complexity of genetic information.\n\nAn increase in the scale of testing available inevitably also means an increase in the range of results returned – many of which, in the current climate, can only be interpreted as ambiguous or uncertain (due to the embryonic nature of our knowledge of this field)9. Such ’Variants of Unknown Significance’ are results where the meaning is unclear and are more likely to be discovered when multiple genes are tested for at once10. In addition to the management of uncertainty, patients will be faced with more information options and so it becomes ever more pertinent to ask ‘how much do you really want to know?’11. Given the ability to look at multiple genes within one test, genomic technologies deliver an opportunity to serendipitously explore genes unrelated to the health condition being explored. This means that when a patient has their cancer genes looked at it would also be possible, at the same time, to explore their genes linked to heart disease. Such results might be referred to as ‘additional looked for findings’3, ‘secondary or incidental findings’ or an ‘opportunistic screen’11. Indeed, within the NHS’s 100,000 Genomes Project parents of a child who is having genome sequencing can have the opportunity to be tested for ‘additional looked for findings’ related to their own risk of future disease, unrelated to their child’s condition12.\n\nGenomic testing is also increasingly being used to guide treatment options and provide more individualised risk assessments, often called ‘precision’ or ‘personalised’ medicine13. This allows clinicians and genetic counsellors to explore predispositions to developing future disease, thereby enabling steps for prevention, screening and/or management to be taken. Furthermore, this is linked to pharmacogenomics – genetic testing used to guide drug use in medicine. For example, before prescribing, patients may be tested first to see if they are likely to be able to metabolise certain drugs14. This is also being used in oncology, where chemotherapies are targeted towards a people with certain genetic profiles15.\n\nGenomic information is different to other sorts of medical information, in that it is shared between biological relatives. So, even if a person is not using healthcare services themselves, they may be related to someone else who is; thus, the reach of the results moves outside of the clinical encounter and into the wider family. Whilst most of us will not currently be a patient in a healthcare setting, we may still be related to someone who is having testing and the questions they have answered may be very relevant to us too. Therefore, the impact of genomic information naturally extends beyond a healthcare encounter–through conversation it travels from the patient, out to their extended family and to people who are not yet patients. Such people could be considered a type of ‘patient in waiting’16; we don’t yet know where and how they seek out information and meaning, but it is likely they look to the media17, popular culture18 and the Internet19 for insight.\n\nThe complex ways that genetics – and now genomics – will affect patients, means that the provision of genetic counselling is of increasing in significance. Patients have voiced the opinion that the provision of genetic counselling is important when having genome sequencing20. Researchers and clinicians have also highlighted the importance of drawing on the expertise of genetic counsellors in order to ensure new technologies are integrated appropriately into healthcare21.\n\nHaving discussed some of the implications of genetics for patients we will now explore some of the ways that genomics will affect people as ‘consumers.’ One way is via ‘direct to consumer’ (DTC) genetic testing. This is a growing industry with private companies marketing and selling a wide range of tests through the Internet. Consumers are able to send off a DNA sample (normally a saliva sample) and a few weeks later receive their test results. There are a wide range of DTC genetic tests both health related and non-health related. Health related tests include identifying predispositions to common and complex disorders, such as cancer and cardiovascular disease, tests for carrier status that could guide reproductive decisions, and nutrigenomics and pharmacogenomics that could potentially guide treatment and lifestyle choices22. Non health-related tests include testing for paternity, ancestry, athletic ability, as well as traits such as earwax characteristics and caffeine metabolism22. DTC genetic testing is rising in prominence. In 2008, 23andMe’s retail DNA testing kit was named invention of the year by Time magazine. In 2016, genomics was named by Forbes as one of the three ‘Big Technologies to Watch’ over the subsequent decade, together with nanotechnology and robotics, predicted to have the biggest impact on society23.\n\nThe potential risks and benefits of DTC genetic testing have been widely debated. Proponents argue that they empower consumers to take responsibility for their health24 and improve the quality of their life25. However, researchers and health professionals have raised concerns about the clinical validity of DTC genetic tests, particularly in relation to susceptibility testing for complex disease, such as diabetes or dementia26,27. Specifically, researchers have argued that these tests fail to take into account non-genetic factors that can contribute to complex disease24. For Mendelian conditions, where a single gene is causative of disease, concerns have been voiced regarding the negative and positive predictive value of DTC genetics tests28. Researchers have also expressed concerns that when testing for serious, potentially life-threatening conditions, there is the possibility for unanticipated emotional reactions that cannot easily be addressed immediately via an Internet based service29. Despite the professional bodies who represent genetic health professionals (e.g. European Society Human Genetics and American College of Medical Genetics and Genomics) recommending that genetic counselling should be provided by DTC genetic testing companies, a review of such services indicated that the support services on offer are often severely lacking30.\n\nDTC genetic testing is a growth market and as genetic testing becomes cheaper an increasing number of online genetic services will be available to members of the public31. With purported plans to incorporate genomic testing into the Apple watch32, as well as fitness monitoring market33, we as consumers will be faced with more opportunities to engage with genomic technology and the implications for our health and wellbeing.\n\nIn making purchasing decisions consumers will increasingly have to draw on their scientific understanding as well as cultural beliefs in order to make purchasing decisions. Abrams and colleagues conclude:\n\n\"Such exposure to genomics information outside of the clinical setting call upon the public to independently evaluate the veracity of these claims and make related decisions\"34.\n\nSo far, we have explored the implications of genetics for us as patients and consumers. Finally, by focussing on some of the novel ethical dilemmas that will arise from technological advancements, we are able to explore some of the broader societal implications of genomics for us as citizens.\n\nScholars have drawn attention to the ways in which commercial companies are exploiting new genomic technologies35 with genomic and post-genomic (e.g. stem-cell) science seen as a crucial aspect of many state economic strategies36. Indeed one of the primary functions of the 100,000 Genomes Project, the NHS genomic sequencing project, is: “Stimulating and enhancing UK industry and investment”12.\n\nThe commercialisation of genomics is inevitable; indeed, to accelerate future drug development, it is imperative. However, with the for-profit industry comes an ethical tension in relation to the raw assets – genomes – that form the resource of data used in genomic research. Empirical research has shown us that whilst publics are willing to donate their DNA and medical information for use in research, they are more suspicious of donating their data for use by for-profit companies37; with concerns that unaffordable medicines would be developed as well as profits made for shareholders that they would not benefit from. We are encouraged as citizens to be altruistic with the donation of our data for the greater good and yet at the same time many will benefit, in a monetary sense, from this altruism. This tension is subject to increasing debate38,39.\n\nGenomic data is commonly stored electronically online; it is exchanged, shared and traded over the Internet every second of the day, as clinicians and researchers and pharmaceutical companies alike explore what a specific variant means. In order to interpret the significance of an individual finding, researchers need to see how this finding has expressed itself previously, by comparing it to datasets from thousands of other people30. The importance of sharing data is outlined by Lucassen and Montgomery (2017) who say:\n\n\n\n“Genomics offers benefits and responsibilities for the individual, the family, the broader community and globally that cannot be realized by keeping the secrets revealed from one genome separate from others.”40(Ch16, p4)\n\nThus, for us as citizens there are now emerging ethical dilemmas in relation to data security and privacy. If the only way to fully realise the potential of genomics is to collect large volumes of genomic data from millions of people (to be accessed by clinicians, non-profit and for-profit researchers in the endeavour to better understand the link between genes and disease), then this means that such data needs to be stored online, shared, and protected against unauthorised use. Together with medical data, the storage of DNA information forms one of the key issues for international policy creation – with the pivotal issue being how to make online data storage safe and secure41.\n\nOne very significant dataset of genomic information is contained within the UK’s National DNA Database. Here, DNA information is stored from people who are convicted, cautioned or recently arrested of a crime and can be matched to DNA collected from crime scenes. Scholars have raised concerns about how DNA should be utilised in criminal law, with DNA databases having a disproportionally high number of people of ethnic minority status42. It was this fact that Lord Justice Stephen Sedley used when arguing for expansion of the National DNA Database. Sedley countered civil liberty concerns arguing that a universal database would be less discriminatory if it was representative of the national population42.\n\nConcerns about discrimination have also been raised in other policy areas regarding genetics. Scholars writing from a disability rights perspective have argued that genetic screening programs can reify a view of disability as purely a medical issue, ignoring social barriers that marginalise people in society. As such, implicit judgments are made about what is a worthwhile life, with genetic screening programmes having the potential to increase discrimination of people with disabilities43.\n\n\nConclusions\n\nIn this opinion article, we have charted some of the wide-ranging implications of advancements in genomics. The implications have been explored in relation to the experience people have in three contexts, as patients, consumers and citizens.\n\nGiven that genomic technology is here to stay and is increasing its foothold across the whole of society, we need to be mindful of taking stock and reviewing what sort of society we want to live in. There needs to be more psychosocial research to understand the attitudes, values and opinions people have about the use and application of genomics. Without this we are in danger of the technology being prescriptive of how society should function, instead of the other way around.\n\nThe societal implications of genomics include many facets. Within this article we provide a brief overview of the availability of genomic tests within medicine and the subsequent increase in engagement with uncertainty, the Direct to Consumer genetic testing market and commercialisation of genomics, the use of DNA databases by the police, the concerns about genomic screening programmes propagating discrimination and the necessity to ensure privacy and security of genomic data. These are all examples of the ways in which genetics/genomics will influence our lives as patients, consumers and citizens, and are all areas that are important for policy consolidation.\n\nGenomic technology is now so pervasive across society and all of us, whether we see ourselves as a patient (or biologically related to a patient), a consumer or a citizen, we are likely to be confronted by the outcomes of genomics. Because of this there is an urgent need to explore the impact of the technology from many different societal perspectives. Together with normative bioethics, empirical data from people making sense of genomics should guide policy decision making so that the implementation of genomic technology is a positive endeavour that benefits humankind.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nAnna Middleton and Jonathan Roberts are supported by the Wellcome Trust [206194]. Jonathan Roberts is funded by the Rosalind Driver Scholarship fund (King’s College London) and by funding from the Public Engagement team at Connecting Science, Wellcome Genome Campus.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to acknowledge Dr Julia Willingale-Theune, Prof. Louise Archer and Dr. Jennifer DeWitt, Prof. Becky Francis and Prof. Justin Dillon for their support in the doctoral research that has influenced this opinion article. From the Wellcome Genome Campus we would also like to acknowledge the support of the public engagement team who have supported the doctoral research project and worked closely with the society and ethics team.\n\n\nReferences\n\nDavies SC: Annual report of the chief medical officer 2016, generation genome. Department of Health. 2017; 1–256. Reference Source\n\nWright CF, Fitzgerald TW, Jones WD, et al.: Genetic diagnosis of developmental disorders in the ddd study: a scalable analysis of genome-wide research data. Lancet. 2015; 385(9975): 1305–1314. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGenomics England: The 100,000 genomes project 2017. 2017. Reference Source\n\nVaithinathan AG, Asokan V: Public health and precision medicine share a goal. J Evid Based Med. 2017; 10(2): 76–80. PubMed Abstract \| Publisher Full Text\n\nCouch FJ, Shimelis H, Hu C, et al.: Associations Between Cancer Predisposition Testing Panel Genes and Breast Cancer. 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N Engl J Med. 2008; 358(2): 105–107. PubMed Abstract \| Publisher Full Text\n\nMiddleton A, Mendes Á, Benjamin CM, et al.: Direct-to-consumer genetic testing: where and how does genetic counseling fit? Per Med. 2017; 14(3). Publisher Full Text\n\nMiddleton A: Your DNA, Your Say. New Bioeth. 2017; 23(1): 74–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHall JA, Gertz R, Amato J, et al.: Transparency of genetic testing services for ‘health, wellness and lifestyle’: analysis of online prepurchase information for uk consumers. Eur J Hum Genet. 2017; 25(8): 908–917. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRegalado A: Apple has plans for your dna. MIT Technology reivew, 2015. Reference Source\n\nHuman K: Maximising your health data. Genome Mag. 2017. Reference Source\n\nAbrams LR, McBride CM, Hooker GW, et al.: The many facets of genetic literacy: assessing the scalability of multiple measures for broad use in survey research. PLoS One. 2015; 10(10): e0141532. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRabinow P, Dan-Cohen T: A machine to make a future: Biotech chronicles. Princeton University Press, 2013. Reference Source\n\nLynch M, McNally R, Atkinson P, et al.: The Handbook of Genetics and Society: Mapping the New Genomic Era. Routledge, 2009. Reference Source\n\nMORI Ipsos: The one-way mirror: Public attitudes to commercial access to health data. London: Wellcome Trust. 2016. Reference Source\n\nParker M, Bull S: Sharing Public Health Research Data: Toward the Development of Ethical Data-Sharing Practice in Low- and Middle-Income Settings. J Empir Res Hum Res Ethics. 2015; 10(3): 217–224. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDove ES, Özdemir V: What role for law, human rights, and bioethics in an age of big data, consortia science, and consortia ethics? the importance of trustworthiness. Laws. 2015; 4(3): 515–540. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLucassen A, Montgomery J, Parker M: Ethics and the social contract for genomics in the nhs. Annual Report of the Chief Medical Officer 2016. Department of Health Chapter 16, 2017. Reference Source\n\nGlobal Alliance for Genomics and Health: White paper: creating a global alliance to enable responsible sharing of genomic and clinical data. 2013. Reference Source\n\nLynch M, McNally R: Forensic dna databases and biolegality. Handbook of Genetics and Society. London: Routledge. 2009; 283–301. Reference Source\n\nShakespeare T: Choices and rights: eugenics, genetics and disability equality. Disability Society. 1998; 13(5): 665–681. PubMed Abstract \| Publisher Full Text" }	[ { "id": "28114", "date": "27 Nov 2017", "name": "Anna Michell", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide an overview of the current state of use of genomic technologies in the UK, comparing this with other nations as well as considering the wider implications of implementing such technologies for clinical practice and everyday life.\nThe use of association data by some companies or tests (rather than direct genomic data) is not dwelt upon in the section on DTC genetic testing although this is highlighted in the referenced literature. The distinction is one which is not always clear to the public and relates directly to the question posed at the end of this section about the lay person's genetic/genomic literacy and how well-equipped they are to evaluate such tests themselves.\nSome consumers also use DTC testing from an Ancestry point of view and do not initially set out to learn medical genetic information from the test. However, tools which put related users in touch with each other can sometimes lead to disclosures of more medically-related information, such as recommendations about screening for inherited conditions.\nThe authors rightly highlight tensions between genomic data donated by patients to research endeavours on an altruistic basis and the possibility of a resultant financial benefit to organisations who provide capital, resources or facilities to support such endeavours. This is juxtaposed with the concomitant lack of value in being unable to share data for comparison and interpretation.\nFinally, they draw attention to the great need for further research into the impact of these technologies on society as a whole - from a variety of perspectives - particularly focusing on the experiences of end-users, be they patients, consumers or citizens. Understanding the needs and concerns of these stakeholders is vital to successful delivery of services in the future.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "28118", "date": "06 Dec 2017", "name": "Lorraine Cowley", "expertise": [ "Reviewer Expertise Genetic counselling", "social science", "bioethics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe scope of this review does not include proof-reading; I have not commented on grammatical or typographical errors.\n\nThe authors have written a timely and relevant opinion piece which should be welcomed by the genetic counselling community. The article aims to explore how we might experience advancements in genomic technology as patients, consumers and citizens and urges policy makers to take stock of the human response to and pervasiveness of genomics. I strongly agree with the authors’ conclusions that genomics is a current and important topic for society and in need of further psychosocial research. However, I would ask the authors to consider further illustrating and discussing the points made in the conclusion throughout the article.\n\nAs a general point, it would be helpful to have a brief statement about the academic disciplinary background of the authors to give the reader a better understanding about the context of the article and an idea about the lens through which their opinions are given.\n\nPerhaps the potted history of the emergence of genomic technology in the abstract would sit better in the introduction with some comment from the authors on how the availability and reduced cost of technology is influencing its application. Some overstatement maybe avoided, for example, the countries they quote where genomic technology is being used outside the UK do not suggest global utilisation and maybe using alternative phrasing such as the ‘economically developed world’ would reflect the situation more accurately. As this is an opinion piece, it would have been good to see more critical engagement with the literature that the authors have cited and more of their opinion. Perhaps each quotation cited could be followed by comment from the authors; whether they contest, agree in part, find problematic or endorse what has been said. It would be helpful to have a brief explanation of their understanding of certain terms and concepts, as there are often inter and intra-disciplinary differences in definitions, for example, it would be helpful to know what their understanding is of ‘normative bioethics’. It is welcome to see mention of the disability rights perspective and issues of potential discrimination with references to social science literature. Given that the opinion piece calls for a societal review, discusses sociological constructs such as citizenship and outlines current applications of genomic/genetic technologies, maybe it would be improved by further engagement with social science literature.\n\nThe authors aim to explore the human response to genetic advancements in genetics. A large proportion of the section headed ‘People as patients’ seems focused more on the technological capacity to offer tests and generate information rather than the human response to those technologies.\n\nThey make the point well about the shared nature of genetic information but stop short of acknowledging that not everyone has the same approach to knowing this information. The point could be enhanced by relating the discussion to the wealth of genetic counselling research that explores the communication of genetic risk in families, thus considering the implications for patients diagnosed with genetic conditions and those who do and do not want to know. I feel that this is a missed opportunity, if not to explore in detail, given the length of the article, at least to mention how finding out genetic information through mainstream diagnostic tests and/or direct to consumer (DTC) genetic testing may influence the communication dynamics within families about genetic risk and what this might mean to people. The authors seem to have assumed normativity in how genetic/genomic information is cascaded throughout families to ‘patients in waiting’ when in contemporary genetic counselling practice we know that this can be complex and challenging.\n\nThe article would be enhanced by either making explicit their stance towards the current and future applications of genomics which they have highlighted, or identifying authors who contest or problematize the issues. For example, but not limited to, it would be interesting to know their opinions on:\n\nDo they uncritically accept that genomics poses responsibilities (Lucassen and Montgomery, 2017) for individuals and what do they understand those responsibilities to be? What place do they think there is for dissenters or test decliners (if any) in the future genomic society? What do they think about mainstreaming? What do they think about the current and purported future use of DTC genetic testing or the issues this currently raises in genetic counselling? What do they make of the claims about the usefulness of genetic information in, for example, the fitness industry? Do they believe that people are equipped to make distinctions about the veracity of claims? Is there a contrasting point to be made in how clinicians interpret the clinical significance of (make distinctions about) genomic information and translate that into clinical care?\n\nThe authors flag the important idea that genomic testing may create uncertainty and this point could be enhanced by critically discussing the ways in which genomics dispels and confirms uncertainty and the limitations of what can be known versus what can be done.\n\nAddressing these issues would go some way to exploring why policy-makers should approach the pervasiveness of genetics with caution. In summary, I believe the authors could state and argue more clearly the limitations to claims that genomics potentially benefits our health, our lifestyle and our cultural practices.\n\nGiven further critical engagement with these arguments, the opinion article could provide a strong overview of nascent genomics in the early twenty first century. This is a really useful article by leading and emerging researchers in the genetic counselling field and it would be beneficial to hear the authors’ opinions more explicitly stated.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "3358", "date": "24 Jan 2018", "name": "Jonathan Roberts", "role": "Author Response", "response": "Thank you very much to Lorraine for her review. We have addressed her comments below (with reviewer comment in italics). As a broad response to the reviewer’s comments we would thank her for thoughtful feedback. We accept that the reviewer draws attention a number of important debates. We would stress that the purpose of this article is to highlight, in a broad way, the myriad implications of genetics (and hence the need for continuing sociological research) without getting drawn into any specific ethical debates in detail. The scope of this review does not include proof-reading; I have not commented on grammatical or typographical errors. The authors have written a timely and relevant opinion piece which should be welcomed by the genetic counselling community. Thank you. While we hope the article will appeal to the genetic counselling community our main hope is that it will be read by a broad audience who may be interested in the future of genetics. The article aims to explore how we might experience advancements in genomic technology as patients, consumers and citizens and urges policy makers to take stock of the human response to and pervasiveness of genomics. I strongly agree with the authors’ conclusions that genomics is a current and important topic for society and in need of further psychosocial research. However, I would ask the authors to consider further illustrating and discussing the points made in the conclusion throughout the article. As a general point, it would be helpful to have a brief statement about the academic disciplinary background of the authors to give the reader a better understanding about the context of the article and an idea about the lens through which their opinions are given. I’m afraid we are restricted to the editorial policies of the journal and so unable to address this. Perhaps the potted history of the emergence of genomic technology in the abstract would sit better in the introduction with some comment from the authors on how the availability and reduced cost of technology is influencing its application. We feel this is covered in the opening paragraph and we believe that an extended history would not be warranted for a short opinion article. Some overstatement maybe avoided, for example, the countries they quote where genomic technology is being used outside the UK do not suggest global utilisation and maybe using alternative phrasing such as the ‘economically developed world’ would reflect the situation more accurately. We agree that it is important not to overstate the implementations of genetics. However, we feel it is important to stress that genomics is a global venture. Indeed the Global Alliance for Genomics and Health, representing 500+ organisations across the world, all involved in some way in the implementation of genomics, including in third world countries. We have added some text to further clarify this on page 2, section “people as patients” paragraph 2. As this is an opinion piece, it would have been good to see more critical engagement with the literature that the authors have cited and more of their opinion. Perhaps each quotation cited could be followed by comment from the authors; whether they contest, agree in part, find problematic or endorse what has been said. It would be helpful to have a brief explanation of their understanding of certain terms and concepts, as there are often inter and intra-disciplinary differences in definitions, for example, it would be helpful to know what their understanding is of ‘normative bioethics’. It is welcome to see mention of the disability rights perspective and issues of potential discrimination with references to social science literature. Given that the opinion piece calls for a societal review, discusses sociological constructs such as citizenship and outlines current applications of genomic/genetic technologies, maybe it would be improved by further engagement with social science literature. We welcome the comments regarding the need for a critical engagement with the literature. The aim and scope of this article was to provide an overview of the wide ranging implications of genomics and as such to draw attention to the need for ongoing research of the type that the reviewer mentions. The article is brief and written for a general audience therefore we do not feel we need to describe what our understanding of certain terminology, e.g. ‘normative bioethics’. The authors aim to explore the human response to genetic advancements in genetics. A large proportion of the section headed ‘People as patients’ seems focused more on the technological capacity to offer tests and generate information rather than the human response to those technologies. We think the reviewer highlights important work, i.e. that which explores the human response to technologies. The aim of this paper was to provide a broad, accessible overview of the implications of genomics and as such to highlight the need for such sociological work. We feel that exploring in detail the sociological literature would be beyond the scope of this paper. They make the point well about the shared nature of genetic information but stop short of acknowledging that not everyone has the same approach to knowing this information. The point could be enhanced by relating the discussion to the wealth of genetic counselling research that explores the communication of genetic risk in families, thus considering the implications for patients diagnosed with genetic conditions and those who do and do not want to know. I feel that this is a missed opportunity, if not to explore in detail, given the length of the article, at least to mention how finding out genetic information through mainstream diagnostic tests and/or direct to consumer (DTC) genetic testing may influence the communication dynamics within families about genetic risk and what this might mean to people. The authors seem to have assumed normativity in how genetic/genomic information is cascaded throughout families to ‘patients in waiting’ when in contemporary genetic counselling practice, we know that this can be complex and challenging. Possible new text: We think the reviewer raises an important point. This paper primarily focuses on the implications of technological advances and the need for ongoing sociological research. However, it is also important to note the rich vein of scholarship, especially that from genetic counselling, that explores the ways people understand and make sense of genetic information. We have added some text - page 3 column 2 paragraph 2 - to draw readers attention to this scholarship. The article would be enhanced by either making explicit their stance towards the current and future applications of genomics which they have highlighted, or identifying authors who contest or problematize the issues. We appreciate the desire for a more explicit stance towards the applications of genomics. However we feel that to do this with such a broad overview of the topic has the potential to triple the length of the paper. As such we have not provided a thorough discussion about additional issues. This being said, the reviewer raises some important points and we have added some text to the paper that acknowledges the authors who have discussed problematic issues that have arisen regarding the implications of genetics. This can be found at page 3, column 1, paragraph 3 For example, but not limited to, it would be interesting to know their opinions on: Do they uncritically accept that genomics poses responsibilities (Lucassen and Montgomery, 2017) for individuals and what do they understand those responsibilities to be? Whether there are indeed responsibilities imposed by genomics and how these are handled by organisations, such as the NHS, could form the basis of a whole new paper and we do not feel we can do justice to this topic within the confines of a sentence or two. What place do they think there is for dissenters or test decliners (if any) in the future genomic society? We have added a sentence to draw readers attention to the important debate with respect dissenters or test decliners.page 3 column 2 paragraph 2, final line. What do they think about mainstreaming? Mainstreaming is such a large topic that it is difficult to summarise this within a sentence or two; we feel this is outside the scope of a broad overview piece. We have also published separately on the emerging roles for genetic counsellors in genomic medicine and the impact of mainstreaming, see Middleton et al What do they think about the current and purported future use of DTC genetic testing or the issues this currently raises in genetic counselling? We have separately published on this issue, see Middleton et al 2016 What do they make of the claims about the usefulness of genetic information in, for example, the fitness industry? Do they believe that people are equipped to make distinctions about the veracity of claims? Is there a contrasting point to be made in how clinicians interpret the clinical significance of (make distinctions about) genomic information and translate that into clinical care? These are all interesting and valid questions. The aim of the paper is to draw attention to the pertinence of these questions, to show the need for continuing research rather than to provide concrete answers or a comprehensive review of the sociological literature. The relationship between different publics, expertise and genetic information is the subject of the author’s PhD and publications detailing this research will be forthcoming. The authors flag the important idea that genomic testing may create uncertainty and this point could be enhanced by critically discussing the ways in which genomics dispels and confirms uncertainty and the limitations of what can be known versus what can be done. Research, especially from genetic counselling, has explored some of the complex ways that patients deal with uncertainty. The potential for increasing levels of uncertainty in genomics provides impetus for further research into how patients cope with, are resilient to and understand uncertainty. We have added some text at page 3, column 1, penultimate paragraph to draw readers attention to the complex debate surrounding uncertainty. Addressing these issues would go some way to exploring why policy-makers should approach the pervasiveness of genetics with caution. In summary, I believe the authors could state and argue more clearly the limitations to claims that genomics potentially benefits our health, our lifestyle and our cultural practices. The reviewer raises important points regarding the potential benefits and limitations of genetics. Within the confines of this short opinions piece we have added texts that further draws attention to this important debate. Given further critical engagement with these arguments, the opinion article could provide a strong overview of nascent genomics in the early twenty first century. This is a really useful article by leading and emerging researchers in the genetic counselling field and it would be beneficial to hear the authors’ opinions more explicitly stated." } ] }, { "id": "28116", "date": "07 Dec 2017", "name": "Michael Arribas-Ayllon", "expertise": [ "Reviewer Expertise Medical sociology and science and technology studies of genetic knowledge", "esp. genetic testing" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a clear and well-structured opinion piece that identifies many relevant issues concerning genetic and genomic testing and its presumed impact on society. I am unfamiliar with this journal, so I am unsure how to gauge the usefulness of this article for its readership.\nMy main concern is the extent to which the authors claim the pervasiveness of genomic technologies. In the sociological literature, with which I am more familiar, these claims are not all clear or straightforward. For instance, Adam Hedgecoe questions the utility of genetic testing, which can increase diagnostic uncertainty (also see Anne Kerr’s work). He also argues that in the context of pharmacogenomics, genomic medicine in the clinic falls short of personalised medicine. Arribas-Ayllon (2016) reviews the literature to show that applications of genetic testing in healthcare are uneven and partial. Sophie Day et al. (2016) has also identified the ways in which genomic medicine further complicates treatment pathways for breast cancer. What is common to these and many other sociological studies is that genomic medicine consistently fall short of the hype that genetic and genomic technologies will be revolutionary and personalizing.\nFor instance, I am troubled by the frequent use of the pronoun ‘all’ in the first paragraph to suggest that genomic medicine is all-encompassing and totalising. It is not at all clear to me that genomic medicine is rolled out as smoothly and as evenly as the authors suggest. In fact, I wonder, given the age of austerity we now live under, whether these technologies will live up to the hype of delivering a ‘truly’ personalised medicine. I also wonder about the circumstances and sites in which this technology is not routinely available to everyone.\nI am also troubled by the assertion made at the end of the third paragraph that genomic medicine ‘has been truly ‘mainstreamed’’. This is a strong statement. What confidence do the authors have that this is actually true? Of course, I think it serves the authors’ argument to confidently state that genomic medicine has arrived and is here to stay because it justifies the urgent need to foster public dialogue about ethical and social implications regarding complex health information. But I feel the authors are overstating these claims. Again, sociological studies maintain that the roll-out of these technologies is not even nor does it not necessarily simplify or improve treatment pathways.\nIn so far as genomic medicine gives out complex and uncertain information, I think the author would benefit from reading the work of Joon-Ho et al. (2013) to elaborate the kinds of issues that arise when ‘returning results’ is framed in terms of a ‘dynamic resource’ of information that needs to be managed over the lifetime of an individual. This also has unique implications for genetic counselling.\nAlthough I understand the authors are discussing genetics in general, the example of DTCGT is based on a different technology to that of the previous examples concerning whole genome sequencing. DTC companies use genotyping to calculate probabilistic risk for common complex disorders. I think the technical differences between sequencing and genotyping ought to be flagged. The general reader might reach the opinion that all genome testing is based on the same technologies.\nIn their Conclusion, the authors claim that there is a danger that genomic technology will be ‘prescriptive of how society should function, instead of the other way around’. Firstly, I don’t think this point has been sufficiently developed in the paper, or at least it doesn’t follow naturally from the arguments and examples given above. I am also concerned that because the author are buying into the revolutionary hype of genomic medicine, that society is construed as being essentially passive.\n\nRecommendations I recommend that the authors tone down their claims that genomic medicine is revolutionary and pervasive. I recommend the authors indicate that there are still significant obstacles of delivering personalised and stratified medicine in real-world settings. I recommend that the authors explore more deeply the kind of relationship individuals can expect from sequence-driven information, especially in relation to how this information is likely to change over time (see Joon-Hu et al. 20131).\n\nMinor revisions: On the last line of the second column on page 2 ‘where chemotherapies are targeted towards a people with…’ should read ‘where chemotherapies are targeted towards people with…’ On page 3, first column, in the second paragraph 2nd line ‘provision of genetic counselling is of increasing in significance’ should read ‘‘provision of genetic counselling is of increasing significance’.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "3357", "date": "24 Jan 2018", "name": "Jonathan Roberts", "role": "Author Response", "response": "Thank you very much to Michael for his review. We have addressed his comments below (Reviewers comments are in italics) This is a clear and well-structured opinion piece that identifies many relevant issues concerning genetic and genomic testing and its presumed impact on society. Thank you. I am unfamiliar with this journal, so I am unsure how to gauge the usefulness of this article for its readership. We chose F1000 because we wanted a platform with good search engine optimisation, so that our article would be very easy to access and find (and thus not be limited by a single journal authorship group). The article has had 364 reads in a matter of weeks, so this bodes well in terms of reaching an interested audience. The paper was written as a broad, light-touch opinion piece as opposed to addressing a specific issue for a particular audience, hence choosing a generic journal that covers all scientific disciplines. We also wrote it for a non-specialist, generic audience. My main concern is the extent to which the authors claim the pervasiveness of genomic technologies. There is absolutely no doubt that genomic technologies are here to stay and are embedded in many different settings. Our reference to the Chief Medical Officers report on Genomics provides evidence to the commitment to further embed genomic technology into healthcare services in the UK. We have also provided evidence (page 3, column 1, ‘people as patients), outside of the UK where genomic technologies are being applied, on a large scale, for example in Australia, Qatar and the USA. In the interests of space we chose not to list the many other global sequencing initiatives, but for the reviewer’s information, we could have added in details about the national sequencing initiatives in Pakistan, Switzerland, Canada, Turkey, Singapore, India, China, Japan and Latin America to name but a few countries to evidence the global reach of genomic technologies. We also draw the reviewer’s attention to the ‘Million European Genomes Project’, where genomic and health data will be linked from 1 million European patients: [Horgan D, Romao M, Hastings R. Pulling the Strands Together: MEGA Steps to Drive European Genomics and Personalised Medicine. Biomedicine Hub. 2017;2(suppl 1)481300 (DOI:10.1159/000481300)]. And also to the work of the Global Alliance for Genomics and Health (www.ga4gh.org) which represents more than 500 organisations worldwide all involved in genomic sequencing initiatives. We have added some text about global utilisation of genomics: “A commitment to significant and ongoing investment in genomics on an international scale is demonstrated by the ‘Million European Genomes Project’, where genomic and health data will be linked from 1 million European patients and The Global Alliance for Genomics and Health (www.ga4gh.org) that represents more than 500+ organisations globally all with the shared endeavour of implementing and enabling genomic technology to be applied in research and clinical practice” In the sociological literature, with which I am more familiar, these claims are not all clear or straightforward. For instance, Adam Hedgecoe questions the utility of genetic testing, which can increase diagnostic uncertainty (also see Anne Kerr’s work). Whilst it is true that the implementation of genomic medicine brings with it many uncertainties (and we have clearly alluded to some of these on page 3, second column, second para) we would contest that that genomic technology is pervasive and being delivered on a large scale. He also argues that in the context of pharmacogenomics, genomic medicine in the clinic falls short of personalised medicine. Arribas-Ayllon (2016) reviews the literature to show that applications of genetic testing in healthcare are uneven and partial. We are not in disagreement with the reviewer about the difficulties that genomic technologies bring and that the application across healthcare settings is uneven. Audiences could be guided to our other publications that offer more of a reflection of what is working and the difficulties, particularly for genetic counsellors, in the application of genomics in a healthcare setting, e.g. : Middleton A, Hall G, Patch C (2015) Genetic counsellors and Genomic Counselling in the United Kingdom. Molecular Genetics and Genomic Medicine 3(2) 79-83.) Instead the purpose of the Opinion piece was to offer a non-specialist audience a summary piece on the different ways genomics will touch us as patients (or related to patients), citizens and consumers. Sophie Day et al. (2016) has also identified the ways in which genomic medicine further complicates treatment pathways for breast cancer. What is common to these and many other sociological studies is that genomic medicine consistently fall short of the hype that genetic and genomic technologies will be revolutionary and personalizing. We don’t disagree with this. But it was not the aim of the Opinion Piece to offer a critique of these issues. Neither was it the aim of the Opinion Piece to provide copious examples of where genomic medicine is working incredibly well (of which there are many). However, it is important to ensure that this article acknowledges the implementation of genomics is complex. As such we have added the following text at page 3, column 1, paragraph 3 “While there are many ways in which genomics is improving healthcare, it is important to note that the implications of genomic medicine are multifaceted. For example Adam Hedgecoe (2008) questions the utility of genetic testing, which can increase diagnostic uncertainty and Sophie Day et al., (2017) has identified the ways in which genomic medicine further complicates treatment pathways for breast cancer. Arribas-Ayllon (2016) reviews the literature to show that applications of genetic testing in healthcare are uneven and partial.” For instance, I am troubled by the frequent use of the pronoun ‘all’ in the first paragraph to suggest that genomic medicine is all-encompassing and totalising. It is not at all clear to me that genomic medicine is rolled out as smoothly and as evenly as the authors suggest. In fact, I wonder, given the age of austerity we now live under, whether these technologies will live up to the hype of delivering a ‘truly’ personalised medicine. I also wonder about the circumstances and sites in which this technology is not routinely available to everyone. In response to the above comment we have ‘toned down’ some of the language. Specifically: In the abstract, paragraph 1, line 8: “‘personalised medicine’ is now a reality” now reads “‘personalised medicine’ is now moving closer to a reality.” In the abstract, paragraph 2, line 2: “The impact of such a ‘genomic era’ is likely to have some level of impact on all of us”, now reads: “The impact of such a ‘genomic era’ is likely to have some level of impact on an increasingly large number of us” I am also troubled by the assertion made at the end of the third paragraph that genomic medicine ‘has been truly ‘mainstreamed’’. This is a strong statement. What confidence do the authors have that this is actually true? In response to this comment some of the language has been changed. In page two, paragraph four, the penultimate sentence “Genomic medicine is now available across whole healthcare systems, it has been truly ‘mainstreamed’” now reads: “As genomic medicine is increasingly available across healthcare systems, we are moving closer to genetics becoming ‘mainstreamed’” While the author raises a valuable point regarding mainstreaming, it is undeniable that genetics is increasingly being used across a range of healthcare settings. For example at the time of writing this comment (17/01/18) research has been published in the Journal of the National Cancer Institute that has provided evidence that BRCA testing for all women over the age of 30 is cost effective. The Society and Ethics Research Group (where the authors are from) are part of the Wellcome Genome Campus in Cambridge, which in turn is a partner in the East of England Genomic Medicine Centre. Through teaching NHS members of staff, funded via Health Education England on the new MSc Genomic Medicine at the University of Cambridge, the authors are in contact with NHS staff involved in ‘mainstreaming’ genomics (i.e. applying new genomic knowledge to their practice). It is this experience that informs our knowledge of the way genomic technologies are influencing practice outside of specialist clinical genetics services. Thus we have confidence and experience in the statements we have made about genomics in a healthcare setting. Of course, I think it serves the authors’ argument to confidently state that genomic medicine has arrived and is here to stay because it justifies the urgent need to foster public dialogue about ethical and social implications regarding complex health information. But I feel the authors are overstating these claims. Again, sociological studies maintain that the roll-out of these technologies is not even nor does it not necessarily simplify or improve treatment pathways. We do not believe the article implied that these technologies are simplified or have improved pathways in all situations. However, there is significant evidence that genomics has improved care in number of settings. Notably, in rare disease and the diagnosis of developmental delay, genomics has made it less likely that patients will have a long ‘diagnostic odyssey’. Importantly for our purposes here, our argument is not based on genomics universally improving care. Rather that genomics will be increasingly used in healthcare settings. This may improve care (such as in rare disease) or may lead to increased uncertainty with a higher likelihood of finding and Variants of Uncertain significance. The key point is that dialogue needs to be fostered because genomics will have increasingly large applications in healthcare, a claim that is well supported through empirical evidence. For examples of genomics improving care see the following reference for details of the cost effectiveness of sequencing in a clinical setting: Walsh et al (2017) The reviewer is also directed to the work of the Deciphering Developmental Disorders project that now has a diagnostic rate of more than 40%, i.e. for children with severe developmental disorders who have not been able to receive a diagnosis for their condition via traditional NHS testing, more than 40% have received their diagnosis after exome sequencing. The DDD project also predicts that if whole exome sequencing was offered to these children as a first line test, more than 50% would have received a diagnosis for their condition : Wright et al (2018) In so far as genomic medicine gives out complex and uncertain information, I think the author would benefit from reading the work of Joon-Ho et al. (2013) to elaborate the kinds of issues that arise when ‘returning results’ is framed in terms of a ‘dynamic resource’ of information that needs to be managed over the lifetime of an individual. This also has unique implications for genetic counselling. We did not want to engage in lengthy theoretical debate about the best ways complexity and uncertainty can be managed in clinical settings (this is a paper in itself!) We have already published our own empirical work (n = 7,000) on the return of results from sequencing studies [see Middleton et al 2016]. We are aware of Joon-Ho Yu et al’s excellent work. However, as this is based on a theoretical approach that is not being applied within clinical settings we feel it is perhaps premature to offer this as a suggestion for how genomic medicine is working in practice now. However, the reviewer draws attention to an important debate. As such we have added the following text at page 3, column 1, penultimate paragraph. . “As patients are exposed to increasingly complex and uncertain genetic information, debates have emerged as to the best way to manage this. Middleton et al., demonstrate that while many people are enthusiastic about receiving genetic information health professions (especially those who work in genetics) are more cautious. Joon-Ho et al., suggest that genetic information should be framed as a ‘dynamic resource’ with uncertainty managed by viewing this information as a resource that should be managed over the lifetime of an individual. There is as yet no consensus regarding the best way to manage the increasing volumes of complexity and uncertainty generated by genetic sequencing. In the ongoing debates we argue it is crucial that debates are informed by wide range of voices.” Although I understand the authors are discussing genetics in general, the example of DTCGT is based on a different technology to that of the previous examples concerning whole genome sequencing. DTC companies use genotyping to calculate probabilistic risk for common complex disorders. I think the technical differences between sequencing and genotyping ought to be flagged. DCT also look for SNPs in genes that cause mendelian disease, the key issue here is their positive/negative predictive value. The general reader might reach the opinion that all genome testing is based on the same technologies. We understand the concern regarding the confusion of what could be called ‘whole genome sequencing’. This term could refer to a wide range to technologies (micro-arrays, SNP arrays, whole exome sequence). We believe we have covered the key concerns of DTCGT in this section (clinical validity for information about common diseases and positive and negative predictive values for mendelian disease). We feel that additional information regarding the specific technologies would not add clarity. However, we have added a short explanation at the beginning on the paper clarifying how we are utilising the term ‘whole genome’ and that this references to a range of different technologies that provide different levels of information. We have added the following text in at the beginning of the article (before “people as patients” section). Before the continuing it is important to make clear that in this article we use the term ‘genomic’ to refer to a wide range of sequencing and laboratory technologies. There are a range of different technologies that interrogate the ‘whole genome.’ Next generation sequencing may be used to create a ‘whole genome sequence’ - a complete sequence all ~ 3 billion base pairs of an individual's genome. However to generate a true whole genome sequence multiple sequencing methods (other than NGS) may need to be used. Furthermore there are a number of technologies that look across an entire genome but do not create a ‘whole genome sequence’ for example, microarrays, SNP arrays, and exome sequences. For our purposes we refer to all technologies that look across the genome as ‘genomic’. Where there are specific technological issues (such as positive and negative predictive values in direct to consumer genetic tests) we refer to these. However, for clarity, we do not feel it it would be beneficial in this article to expand on the different technologies used for sequencing in different settings. As such for purposes of this article we discuss broadly the implications issues raised by ‘genomic’ technology with an understanding that this refers to a range of sequencing and testing techniques. In their Conclusion, the authors claim that there is a danger that genomic technology will be ‘prescriptive of how society should function, instead of the other way around’. Firstly, I don’t think this point has been sufficiently developed in the paper, or at least it doesn’t follow naturally from the arguments and examples given above. I am also concerned that because the author are buying into the revolutionary hype of genomic medicine, that society is construed as being essentially passive. The point is to highlight the fact that different publics’ views need to be taken in account as genomic technology becomes increasingly relevant in a range of settings. Whilst we would not want to construe the public as passive, we must not ignore the importance of power relationships and how in discourses of scientific and medical expertise ‘non- expert’ voices can be ignored. We believe that while it is important to value expertise, we must also ensure that there is dialogue and that non-expert voices do not become marginalised. This is why research into the attitudes and opinions of different publics is so valuable. We would also point out that there are empirical examples of how new technologies can become prescriptive of the way society functions. This happens when what can be done is conflated with what should be done. For example, with prenatal screening programs, there is evidence that as new technologies become routinised they have driven medical practice. This happens when the ‘normal’ thing to do has became conflated with the ‘right’ thing to do. Scholars writing from a disabilities rights perspective have noted that this may subvert the wishes of patients who may not want screening. In a medical settings - where there are powerful power dynamics in play - patients may not feel empowered to state a preference that goes against what is ‘normal’ and ‘right’. It is this we refer to when we say that technology may become prescriptive and we have added some text to further this argument on page 5, paragraph 2 of ‘conclusions’ section. Recommendations I recommend that the authors tone down their claims that genomic medicine is revolutionary and pervasive. We hope we have managed to re-defend our view that genomics is indeed pervasive across society. We have, however, toned down our claims as requested. I recommend the authors indicate that there are still significant obstacles of delivering personalised and stratified medicine in real-world settings. We have added reference to the obstacles to delivering personalised medicine, however, we have not elaborated in detail on these as this wasn’t the focus of the Opinion Piece and we wanted to keep the piece deliberately broad. I recommend that the authors explore more deeply the kind of relationship individuals can expect from sequence-driven information, especially in relation to how this information is likely to change over time (see Joon-Hu et al. 20131). We feel this could form a detailed discussion in a different paper (postulating on the possible futures that could exist with different applications of genomic medicine); however, Joon-Hu et al’s theoretical debate about the relationship between person and their data, is not being applied in practice at the moment and thus we do not feel it should form a significant part of our current Opinion Piece. Minor revisions: On the last line of the second column on page 2 ‘where chemotherapies are targeted towards a people with…’ should read ‘where chemotherapies are targeted towards people with…’ Corrected On page 3, first column, in the second paragraph 2nd line ‘provision of genetic counselling is of increasing in significance’ should read ‘‘provision of genetic counselling is of increasing significance’. Corrected" } ] }, { "id": "28119", "date": "07 Dec 2017", "name": "Tina-Marie Wessels", "expertise": [ "Reviewer Expertise Genetic counselling" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis opinion piece covers the pertinent issues we are currently grappling with as genomic medicine gains momentum. The authors highlighted the many facets and far-reaching consequences well.\nThe authors discussed how we have moved from single genes to panels (many genes) to whole exome and genome sequencing and the use of bioinformatics to identify relevant variants. An important issue is the different tools available in bioinformatics, how these are applied in specific cases and how these effect what is regarded as significant and what is not. This field is growing and it will continue to improve its algorithms so to increase the accuracy of variant calling. In addition, distinguishing between normal variation and pathogenicity is currently problematic when working with non-European populations. Concerted affords are underway to address the underrepresentation of diverse population (Ramesar 20151).\nThe authors state that genetic counselling will be increasingly more significant. It is important to mention that the role of clinical genetics i.e. taking a comprehensive family history and using clinical information to guide and or assist with the interpretations of genomic data will remain important. Except for the traditional role of genetic counsellors helping consumers understand and make informed choices as pointed out, the role of genetic counsellors is expected to expand to include providing support to other healthcare providers involved with the ordering of genomic tests, assistance in developing online tools and education for both professionals and the public.\nOne aspect that requires more consideration in the genomics era is the need for education. The authors quote Abrams and colleagues that highlight the need for public education so that consumers can make informed decisions. Education for the public is essential but there is also a need for education of medical and other health care professionals as they will increasingly be involved in ordering and or interpreting genomic tests and use these results in the care of their patients. There are so many tests to choose from which makes it difficult to evaluate which have clinical utility.\nThe authors highlighted the ethical dilemmas related to data security and privacy. Ethical dilemmas have been highlighted since the start of the human genome project (ELSI). The issues related to incidental findings and informed consent could be included in the article.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2020
https://f1000research.com/articles/7-102/v1	23 Jan 18	{ "type": "Research Article", "title": "A pilot study comparing pattern of damage sustained among instruments from different surgical units in a tertiary care centre in Nepal – reappraising the role of instrument reprocessing in retaining their value", "authors": [ "Sunil Munakomi", "Roshan Shah", "Sangam Shrestha", "Roshan Shah", "Sangam Shrestha" ], "abstract": "Background: The quality of instruments plays a pivotal role in governing safe operating room culture. The reprocessing system followed in the institution determines their durability thereby ensuring patient safety as well as minimizing health spending. Rigorous reprocessing in a centralized instrument reprocessing department by well trained staff following formulated guidelines helps to achieve the target of “safe surgery saves lives” as formulated by the World Health Organization. Methods: We sought to determine the patterns of wear and tear sustained among sets of surgical equipment from two surgical units that had been sent to the repair department within a year of their purchase. Analysis of similar changes in the joints of the instrument, as well as pattern of fractures sustained was performed. Results: All patterns of wear and tear were common in both the general surgical arm and neurosurgical counterpart, with the exception of fractures and mal-alignments. Similar study was performed examining changes in the joints. Stains were the most commonly observed change pattern in both sets of instruments. Fractures were most frequent in the working ends in both sets of instruments. Conclusion: There is an alarming incidence of wear and tear patterns in the instruments used in the surgical units, even within the first year of their use. This supports the strict implementation of reprocessing guidelines by well trained workers and their quality assessments via audit checks. The quality of the purchased instruments also plays a pivotal role.", "keywords": [ "surgical", "instruments", "damage", "pattern", "reprocessing" ], "content": "Introduction\n\nSurgical instruments are important assets to any surgical unit. The quality of this armamentarium links to the smooth running of operating theatres, as well as ensuring patient safety (see presentation on surface changes in surgical instruments here). The quality of their reprocessing minimizes the ‘wear and tear’ process, thereby ensuring their durability. It is paramount that formulated guidelines on the methods of reprocessing and sterilizing surgical instruments are implemented1. This is more prudent in developing nations with limited resources, as it can help minimize their health costs. Simple steps of wiping out surgical soils (blood) from the instruments, use of demineralized water while rinsing, and proper storage contributes to better durability and improved patient safety. We, hereby, perform a pilot study focusing on the pattern of damage sustained in surgical instruments to determine the efficiency of instrument processing quality and instrument handling among different surgical units. This study aims to help reappraise the role of proper reprocessing and handling of surgical instruments, which is not well prioritized in the developing nations with limited resources. This would maintain the value of the instruments thereby increasing the durability and minimizing costs.\n\n\nMethods\n\nThis study was carried out to determine the patterns of damage sustained among the instruments sent to the instrument maintenance unit of the Nobel Teaching Hospital, Nepal, from its different surgical units. Different sets of instruments were studied examining the pattern of changes seen with regards to spots, stains, pits, cracks, fractures, rust, mal-aligned parts, and tightening and loosening of the joints. Equal sets of instruments (47 each on comparable basis to the total of 47 neurosurgical instruments included in this study) sent to the institutional instrument maintenance unit from the departments of General Surgery and Neurosurgery, citing their repair or replacement within a year of their use, were analyzed. A comparative study was then conducted analyzing patterns in the ‘wear and tear’ process sustained among the instruments to provide some perspective on the quality of the reprocessing within the institution (see AKI brochure on instrument reprocessing techniques) (Figure 1–Figure 3).\n\nThe role of cleaning, handling and storing and the subsequent damages sustained among the instruments from the two surgical units was then studied. Further to this, a comparison of similar changes seen within the joints of the instruments from these departments was performed. The joint was chosen because of the high friction during usage, and the propensity for retained surgical soils within them, which predisposes to corrosion, stains, cracks and fractures. This was considered the signature marker of the quality of cleaning and reprocessing. Lastly, differences in the location of the fractures on the instruments was also documented (Figure 4).\n\nThis study was cleared by the Institutional Review Committee of Nobel Teaching Hospital, Biratnagar, Nepal.\n\n\nResults\n\nStains was the most common observation, seen in 97.87% of the instruments, followed by loosening in 82.97%, rust in 27.65%, pits in 25.5% and mal-alignment in 19% of the instruments (Figure 5).\n\nWhen assessing the joints, loosening was seen in 82.97%, stains in 80.85%, rust in 29.78% and tightening in 14.89%of the instruments (Figure 6).\n\nFractures were seen in 17.02% of the instruments, mostly found at the tip (75%) followed by involvement of the shaft and handle (12.5% each).\n\nStains were the most common observation, found in 38.29% of instruments, followed by loosening in 31.91%, mal-alignment in 29.78% and discoloration in 23.4% (Figure 7).\n\nWhen assessing the joints, loosening and staining were seen in 29.78% each, whereas rust was found in only 8.5% (Figure 8).\n\nFractures were seen in 27.65%, mostly at the tip (46.1%), followed by involvement of the joint in 23% and shaft and handle in 15.38% each.\n\nComparing the patterns of damage among the instruments from the two different surgical units, all patterns were higher in the general surgical arm, except for the incidence of fractures (27.65% vs 17.02%) and mal-alignment (29.78% vs 19%) (Figure 9).\n\nArrows indicate where the incidence of a specific change is higher in neurosurgery compared to general surgery.\n\nExamining the damage in the joint areas, more damage was seen in the instruments from the general arm with the exception of fractures sustained (6.3% vs 0%) (Figure 10).\n\nArrows indicate where the incidence of a specific change is higher in neurosurgery compared to general surgery.\n\nThe most common site of fractures in instruments from both units was in the tip (75% vs 46.1%).\n\n\nDiscussion\n\nSpaulding classified instruments into critical, semi-critical and non-critical depending upon the risk involved in transmitting infections through their usage2. Recent studies have proven the presence of significant amount of stains, soils, structural damage as well as bio-films in ready to use instruments despite multiple stages of processing3. Despite the recent advancements made in the medical field, infection transmission following improper reprocessing is still a major concern4,5. Proper processing of instruments therefore ensures better durability and improves patient safety. With this in mind, the reprocessing unit should precisely follow the manufacturer's instructions and the relevant institutional guidelines (see presentation on surface changes in surgical instruments here). It is therefore, prudent to establish, standardize and audit the guidelines on reprocessing of medical devices6. This ensures better safety for patients and health workers as well as enhancing the value retention of the instruments (see presentation on surface changes in surgical instruments here).\n\nRecent guidelines recommend implementing compliance with set protocols in a Central Sterile Supply Department (CSSD), with trained staff, in a controlled environment. The whole process should be periodically counter checked and well audited6. All steps involved, such as disassembling, sorting, soaking, cleaning, rinsing, drying, reassembling, inspecting, lubricating, and wrapping, should be properly implemented6.\n\nThe Instrument Reprocessing Working Group (AKI) has recently formed a red brochure that details all damages that can occur during processing and sterilization, as well as the means to minimize them (see AKI brochure on instrument reprocessing techniques). Impurities in the rinsing water can lead to stains (silicates), pitting (chlorides), and blackening (ammonium ions). Improper care of the joints of the instruments can lead to corrosion, cracks and thereby facilitate early fractures. Inadequate drying can lead to rusting in the instruments. The use of demineralized water, proper cleaning, and avoiding dampness ensures better protection from spots, stains, rusts and pits, thereby increasing their durability and minimizing health costs (see AKI brochure on instrument reprocessing techniques). Proper handling, correct loading, ensuring material compatibility, timely lubrication (milking) and periodic checks for surface integrity further ensure their maintenance. They have further recommended a maximum waiting period of 6 hours between usage of instruments and start of cleaning, and as well as usage of demineralized water in the reprocessing cycle (see AKI brochure on instrument reprocessing techniques).\n\nOur study showed that stains were the most common changes seen in the instruments. This suggests either improper wiping off of soils or the deposition of impurities in the rinsing processes. Low incidence of such changes in the neurosurgical unit indicates a better reprocessing attitude in the unit. However, better quality checks of the rinsing water for impurities, and better clearing of the surgical soils retains paramount value in reprocessing cycle. The high incidence of fractures and mal-alignment in the neurosurgical sets may be attributable to the frequent usage of fine micro-instruments with sharp working ends with fine joints and springs in the department. Better care of such fine and expensive equipment may also have resulted in them faring better in comparison to the general surgery counterpart. The high incidence of stains rusts and loosening seen among the instruments from the general surgery units may be attributable to their long usage, and also their sharing among different units before the procurement of new sets. Instruments in neurosurgery are used by limited surgeons and there is a propensity for faster replacement, owing to the damage to the fine working ends. Chronic changes such as rust, cracks and corrosions are observed less in the neurosurgical unit. One of the limiting factors of our study can be the quality of the instruments purchased in the departments. However, any surgical instrument is said to have shelf-life of at least 10 years if adequate care of the instruments is taken7.\n\nThe World Health Organization (WHO) has already implemented the notion of “Safe Surgery Saves Lives”, and formulated strict guidelines governing them8. Safe operating room culture can prevent sentinel events that can prove to be devastating as well as help minimize malpractice crisis9. The ‘cleanability’ and configuration of instruments bears immense impact upon the patient safety10,11. The Joint Commission found 74% of all “immediate threat to life declarations” were directly related to improperly sterilized instruments. They therefore formulated a checklist to minimize this (see AKI brochure on instrument reprocessing techniques). One study has shown major concerns regarding the effectiveness of sterilization being practiced in low and middle income countries12. Despite the prevalence of such poor practices in developing countries, proper assessment tools can eliminate these loop-holes13. A curriculum based on patient safety needs to be addressed and practiced for ensuring patient safety14. Such practices promote safe surgical practice15,16. This also prevents the formation of bio-films on the surfaces of surgical instruments17,18. There is utmost need for a paradigm shift in sterile processing departments, and the implementation of new approaches in processing19,20. This requires acquisition of a thoroughly trained workforce for the process21. Paradoxically unqualified workers are more often placed in these roles, thereby jeopardizing the whole reprocessing cycle22. Furthermore, continuous and systematic quality improvement monitoring needs to be implemented23.\n\n\nConclusion\n\nProper instrument handling and their reprocessing needs be of primary importance. Strict guidelines need to be formulated and strictly implemented in a centralized area in a dogmatic fashion. This is even more prudent in the context of resource limited setups. This culture promotes the practice of safe surgery and thereby maximizes patient’s safety. Our study also highlighted the fact that simple steps in the reprocessing such as cleaning, handling, wrapping and storage of surgical instruments can have significant impact on the overall durability of the instruments, which are the working hands of any surgeons. Care also needs to be given on purchasing quality instruments and also for allocating trained manpower for this critical reprocessing cycle. Such practice can minimize health investments thereby allowing improved allocation of limited health resources available in developing countries like ours. This would also help minimize the malpractice crisis that is slowly lurking in the health sector in the global front.\n\n\nData availability\n\nDataset 1: Tables listing all observed patterns of damage for each instrument surveyed in the study 10.5256/f1000research.13699.d19091024.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nCanadian Standards Association: CAN/CSA-Z314.0-13 Medical Device Reprosessing - General requirements. Rexdale, Ont.: Canadian Standards Association; 2013. Reference Source\n\nSpaulding E, editor : The role of chemical disinfection in the prevention of nosocomial infections. International Conference on Nosocomial Infections; 1970 1971; Chicago, IL: American Hospital Association.\n\nde Melo Costa D, de Oliveira Lopes LK, Tipple AF, et al.: Evaluation of stainless steel surgical instruments subjected to multiple use/processing. Infection, Disease & Health. (Ahead of print). Publisher Full Text\n\nLipscomb IP, Sihota AK, Keevil CW: Comparative Study of Surgical Instruments from Sterile-Service Departments for Presence of Residual Gram-Negative Endotoxin and Proteinaceous Deposits. J Clin Microbiol. 2006; 44(10): 3728–3733. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDancer SJ, Stewart M, Coulombe C, et al.: Surgical site infections linked to contaminated surgical instruments. J Hosp Infect. 2012; 81(4): 231–8. PubMed Abstract \| Publisher Full Text\n\nBest Practices for Cleaning, Disinfection and Sterilization of Medical Equipment/Devices In All Health Care Settings, 3rd edition. 2013. Reference Source\n\nSpry CC: Care and Handling of Basic Surgical Instruments. AORN J. 2007; 86: S77–S81. Publisher Full Text\n\nWorld Health Organization: Safe surgery saves lives. 2013. Reference Source\n\nMakary MA, Sexton JB, Freischlag JA, et al.: Patient Safety in Surgery. Ann Surg. 2006; 243(5): 628–635; discussion 632–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChobin N, Swanson K: Putting Patient Safety First: The Sterile Processing Department and Healthcare Technology Management. Biomed Instrum Technol. 2012; 46(Suppl): 27–31. PubMed Abstract \| Publisher Full Text\n\nScheinberg N, Zhang B, Raschid L, et al.: A Systematic Approach to Improve the Reprocessing of Surgical Instruments. In: Duffy V, Lightner N. (eds) Advances in Human Factors and Ergonomics in Healthcare. Advances in Intelligent Systems and Computing, Springer, Cham, 2017; 482. : 275–286. Publisher Full Text\n\nO’Hara NN, Patel KR, Caldwell A, et al.: Sterile reprocessing of surgical instruments in low- and middle-income countries: A multicenter pilot study. Am J Infect Control. 2015; 43(11): 1197–200. PubMed Abstract \| Publisher Full Text\n\nFast O, Fast C, Fast D, et al.: Limited sterile processing capabilities for safe surgery in low-income and middle-income countries: experience in the Republic of Congo, Madagascar and Benin. BMJ Global Health. 2017; 2(Suppl 4): e000428. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim FJ, da Silva RD, Gustafson D, et al.: Current issues in patient safety in surgery: a review. Patient Saf Surg. 2015; 9: 26. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeavey R: High-level disinfection, sterilization, and antisepsis: current issues in reprocessing medical and surgical instruments. Am J Infect Control. 2013; 41(5 Suppl): S111–S117. PubMed Abstract \| Publisher Full Text\n\nSeavey RE: Safe instrument reprocessing: the perioperative role. AORN J. 2015; 101(4): 482–5. PubMed Abstract \| Publisher Full Text\n\nGuideline for cleaning and care of surgical instruments. In: Guidelines for Perioperative Practice. Denver, CO: AORN, Inc; 2015; 615–650.\n\nAssociation for the Advancement of Medical Instrumentation: ANSI/AAMI ST79: Comprehensive guide to steam sterilization and sterility assurance in health care facilities. Arlington, VA: Association for the Advancement of Medical Instrumentation; 2013. Reference Source\n\nSwanson SC: Shifting the sterile processing department paradigm: a mandate for change. AORN J. 2008; 88(2): 241–7. PubMed Abstract \| Publisher Full Text\n\nBlyth PL, Shimkus J: New tools need new approaches. The artistry of SPD (sterile processing department). Mater Manag Health Care. 2000; 9(7): 24–5. PubMed Abstract\n\nGarner M, Collins A, Dunphy S, et al.: Developing a central sterile surgical instrument technician program. AORN J. 2004; 80(2): 235–8, 241. PubMed Abstract \| Publisher Full Text\n\nTipple AF, de Souza TR, Bezerra AL, et al.: [The worker with no training in nursing in a center of materials and sterilization: a challenge to the nurse]. Rev Esc Enferm USP. 2005; 39(2): 173–180. PubMed Abstract \| Publisher Full Text\n\nChoo S, Papandria D, Goldstein SD, et al.: Quality improvement activities for surgical services at district hospitals in developing countries and perceived barriers to quality improvement: findings from Ghana and the scientific literature. World J Surg. 2013; 37(11): 2512–9. PubMed Abstract \| Publisher Full Text\n\nMunakomi S, Shah R, Shrestha S: Dataset 1 in: A pilot study comparing pattern of damage sustained among instruments from different surgical units in a tertiary care center in Nepal – reappraising the role of instrument reprocessing in retaining their value. F1000Research. 2018. Data Source" }	[ { "id": "30137", "date": "06 Feb 2018", "name": "Vikal Chandra Shakya", "expertise": [ "Reviewer Expertise General and gastrotintestinal surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is well-written with comprehensive review of the literature; with good conclusions; with definitely a contribution to practice of the operating surgical units. Good evaluation of the results is another best aspect of this article. The conceptualization and analysis is commendable with well written English. This article would definitely support assessment of the the purchased instruments to be applied in the future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "38035", "date": "26 Sep 2018", "name": "Samer S. Hoz", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article represents a brief and concise review. The topic under focus “surgical instruments-care improvement” is an interesting topic and contributes valuable added knowledge to the field. It is written in an accepted language skill with consistent attention to the needs of readers in a clear and comprehensive organization. The strength of the article is that it presents a clear summation of a critical daily surgical concerns. Its weakness may be that it does not offer adequate solutions rather than highlighting the different forms and patterns of damages. In summary, the central concepts clearly defined within the article with adequate evidence, fits the journal field and contributes to advance the knowledge in the surgical field.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-102
https://f1000research.com/articles/7-101/v1	23 Jan 18	{ "type": "Opinion Article", "title": "UTAUT for HSS: initial framework to study health IT adoption in the developing countries", "authors": [ "Anis Fuad", "Chien-Yeh Hsu", "Chien-Yeh Hsu" ], "abstract": "Unified Theory of Acceptance and Use of Technology (UTAUT) is an integrative concept that has been used widely to measure IT adoption. However, a recent study in a developing country concluded that UTAUT is not adequate in predicting IT adoption within the context of health system strengthening (HSS). It has been suggested that context-specific dimensions to modify UTAUT should be considered. The objective of this paper is to propose an extension of the theory, called UTAUT for HSS, as a reference for contextualizing health system variables for health IT adoption studies in the developing countries. We combined the multi-level framework of UTAUT with WHO health system building blocks. Modification of the original multi-level framework was performed on the 3 levels. i.e: the higher-level contextual factors, middle-level, and individual-level contextual factors. Based on this, we propose a modified multi-level framework of technology acceptance and use for health system strengthening setting (UTAUT for HSS). Given the complexities of health systems, more thoughts regarding the methodologies will be useful to enrich this initial framework. Commentaries and discussions are invited for improvement, before implementation to obtain more complete story of health IT adoption in the low resources setting.", "keywords": [ "UTAUT", "health system strengthening", "developing countries" ], "content": "Introduction\n\nImproved health status of the population can only be achieved by a strengthened health system. Health system strengthening (HSS) efforts require a sound and reliable health information system1. Consequently, adoption of information technology (IT) becomes inevitable to improve data use, program management, and health services. Despite the high expectation, adopting IT in a health system is far from simple. Similar interventions can produce opposite results in different health system environments. From non-adoption and abandonment on one side to a well adopted and sustainable use in another side2. Multiple factors, individual to social, are involved and interact each other influencing the behavior to adopt the technologies.\n\nUnified Theory of Acceptance and Use of Technology (UTAUT) is an integrative concept that has been used widely to measure IT adoption since the original paper introduced in 20033. UTAUT formulated four core determinants (performance expectation, effort expectation, social influence, and facilitating conditions) and up to four moderators (gender, age, experience, and voluntariness of use) influencing individual behavior to adopt and use IT. Scopus scholarly database recorded 8939 citations to UTAUT as of 16 January 2018. Although this has been cited in many fields for more than a decade, nevertheless, only a limited number of papers contributed to integration of UTAUT with external theories or extend the original conceptual framework4.\n\nUntil 16 January 2018, in PubMed, about 106 articles specifically mentioned UTAUT. Those studies were conducted in developed and developing countries. A recent study from Cameroon where public hospitals clinicians adopted hospital information systems, provided an interesting insight. The authors concluded that UTAUT is not adequate in predicting health IT adoption in a developing country setting. For researchers aiming to measure health IT adoption in developing countries, they suggested considering context-specific dimensions in modifying UTAUT5.\n\nThis paper aims to contextualize health system dimensions to study IT adoption using UTAUT framework in the low-resources setting. We try to integrate UTAUT with health system frameworks of WHO1 with an expectation to invite more debates regarding the appropriate models to evaluate health IT intervention for health system strengthening initiatives, particularly in the developing countries.\n\n\nUTAUT for HSS: extension of UTAUT for health system strengthening\n\nCognizant of the low number of UTAUT theoretical integration or extension research, UTAUT authors structured four promising extensions of UTAUT. These include new exogenous mechanisms, new endogenous mechanisms, new moderation mechanisms, and new outcome mechanisms. New exogenous mechanisms represent the impacts of external predictors to the four core determinants (i.e., performance expectation, effort expectation, social influence, and facilitating conditions). New endogenous mechanisms could include: 1) new predictors’ impact on the two endogenous variables (i.e., behavioral intention and use behavior) or 2) the enhancement of the four core determinants and the two endogenous variables. New moderating mechanisms involve new moderating effects complemented to the original UTAUT. New outcome mechanisms refer to the new effect of behavioral intention and technology use. All the four extensions are integrated into a multi-level framework consisting of higher-level contextual factors, a middle level containing the baseline model of UTAUT, and individual level contextual factors4.\n\nThis new framework offers opportunities to contextualize system-wide variables within the health system building blocks as predictors influencing the status of health IT adoption. Characteristics of developing countries (in national, sub-national or service delivery level) are very relevant to be embedded in the framework. A recent study identified four contextual factors in developing countries, namely hierarchical roles, aid funding, corruption, and competing priorities that potentially influence the success of health information strengthening6. Developing countries also face a shortage of health workers in rural areas, the variable quality of care, lack of patient compliance, and fraud7.\n\nThe above issues can be classified according to the six health building blocks i.e.1) service delivery, 2) health workforce, 3) information, 4) medical products, vaccines & technologies (MPVT), 5) financing, and 6) leadership and governance1. Progress and characteristics of the information building block in a country depend on the following components: 1) leadership and governance, 2) strategy and investment, 3) services and applications, 4) standards and interoperability, 5) infrastructure and 6) workforce8.\n\nTherefore, we propose UTAUT for HSS, a modified multi-level framework of technology acceptance and use for health system strengthening setting (Figure 1). We combined the UTAUT multi-level framework4 with WHO health systems building block1 to address the issue of contextualizing health system variables to improve health IT adoption models. Compared to the original framework4 we performed a modification at the three levels. In the higher-level, we have changed the original attributes (environment, organization, and location) with five health system building blocks. In the middle-level, we have changed the original new outcome phenomenon with three related outcomes namely individual (health workforce) outcome, intermediate (health system) outcome and overall (health system) outcome. In the individual-level, we specifically mention health workforce and health IT attributes. E-health building blocks as proposed by WHO-ITU8 could be embedded into the existing health system building blocks.\n\nAnother health IT research in a developing country reported process, result, and policy implication of electronic medical record (EMR) readiness assessment on 381 health facilities in Kenya9. This report is an example of event-time related activities before the implementation of health IT intervention. Since IT adoption is a time-dependent process, this current framework allows for multiple assessments periodically that could be compared to measure the progress of adoption.\n\nAlthough promising, researchers wanting to use UTAUT in HSS setting should consider the potential limitations. Many UTAUT studies depend on a restricted user type, such as the office workers. In health system context, involving a different type of user including patients, various type of health workers and policymakers will enrich the big picture. UTAUT has long been recognized as a functionalist-institutionalist theory that focuses on systemic stability. Combining with other interpretive theories will be useful to better understand the complex social and symbolic reality of health IT adoption10. This will unsurprisingly trigger the discussion of the pro and con of using other methods such as Implementation Research, Mixed Methods, and Qualitative Studies to apply UTAUT within the milieu of HSS setting.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work\n\n\nReferences\n\nWorld Health Organization: Everybody's business--strengthening health systems to improve health outcomes: WHO's framework for action. 2007. Reference Source\n\nGreenhalgh T, Wherton J, Papoutsi C, et al.: Beyond Adoption: A New Framework for Theorizing and Evaluating Nonadoption, Abandonment, and Challenges to the Scale-Up, Spread, and Sustainability of Health and Care Technologies. J Med Internet Res. 2017; 19(11): e367. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVenkatesh V, Morris MG, Davis GB, et al.: User acceptance of information technology: Toward a unified view. MIS Quarterly. 2003; 27(3): 425–478. Publisher Full Text\n\nVenkatesh V, Thong JY, Xu X: Unified Theory of Acceptance and Use of Technology: A Synthesis and the Road Ahead. J Assoc Inf Syst. 2016; 17(5): 328–376. Reference Source\n\nBawack RE, Kala Kamdjoug JR: Adequacy of UTAUT in clinician adoption of health information systems in developing countries: The case of Cameroon. Int J Med Inform. 2018; 109: 15–22. PubMed Abstract \| Publisher Full Text\n\nThomas JC: Contextual factors affecting health information system strengthening. Glob Public Health. 2017; 12(12): 1568–1578. PubMed Abstract \| Publisher Full Text\n\nLewis T, Synowiec C, Lagomarsino G, et al.: E-health in low- and middle-income countries: findings from the Center for Health Market Innovations. Bull World Health Organ. 2012; 90(5): 332–340. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWorld Health Organization, International Telecommunication Union: National eHealth strategy toolkit. 2012. Reference Source\n\nMuthee V, Bochner AF, Kang'a S, et al.: Site readiness assessment preceding the implementation of a HIV care and treatment electronic medical record system in Kenya. Int J Med Inform. 2018; 109: 23–29. PubMed Abstract \| Publisher Full Text\n\nSovacool BK, Hess DJ: Ordering theories: Typologies and conceptual frameworks for sociotechnical change. Soc Stud Sci. 2017; 47(5): 703–750. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "36269", "date": "27 Jul 2018", "name": "Vijayan Kumara Pillai", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article makes valuable contribution to the theory of UTAUT. Even though the extension suggested is by no means surprising, the authors have explained it well and conveyed their ideas both diagrammatically and in words. Only criticism is that, they have not reported any empirical support for the theoretical extension proposed.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3866", "date": "02 Aug 2018", "name": "Anis Fuad", "role": "Author Response", "response": "Thanks for your useful comments and advice. We will elaborate examples of the empirical evidence in the second version of this paper." } ] }, { "id": "36284", "date": "30 Jul 2018", "name": "Eric D. Perakslis", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article would benefit from the inclusion of definition and context for health information technology. For example, by HIT, does the author describe full EMR implementations, which are quite complex, versus other HIT tools? Is the proposed framework suitable and needed to all forms of HIT efforts?\nSecond, the author briefly describes other methodologies at the end of the paper. It would be helpful to consider a few other methods in a bit more detail to compare and contrast the potential of the proposed/modified UTAUT approach.\nLastly, the piece could benefit from examples of the proposed recommendations, even if hypothetical, that would provide some practical grounding and understanding of the proposed enhancements to the UTAUT framework.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3867", "date": "02 Aug 2018", "name": "Anis Fuad", "role": "Author Response", "response": "We thank you for the constructive comments and suggestions to this paper. We agree to elaborate on the definition of health IT, comparisons with other methodologies and hypothetical examples that will come up from this framework. We will accommodate those issues in the second revision of this paper." } ] } ]	1	https://f1000research.com/articles/7-101
https://f1000research.com/articles/7-100/v1	23 Jan 18	{ "type": "Research Article", "title": "Comparative genomic analysis of crustacean hyperglycemic hormone (CHH) neuropeptide genes across diverse crustacean species", "authors": [ "Wai Hoong Chang", "Alvina G. Lai", "Wai Hoong Chang" ], "abstract": "Background: Recent studies on bioactive peptides have shed light on the importance of these compounds in regulating a multitude of physiological, behavioral and biological processes in animals. Specifically, the neuropeptides of the crustacean hyperglycemic hormone (CHH) superfamily is known to control a number of important functions ranging from energy metabolism, molting, osmoregulation to reproduction. Methods: Given the importance of this peptide family, we employed a conservative approach utilizing extant transcriptome datasets from 112 crustacean species, which not only include important food crop species from the order Decapoda, but also from other lower order crustaceans (Branchiopoda and Copepoda), to identify putative CHH-like sequences. Results and conclusions: Here we describe 413 genes that represent a collection of CHH-like peptides in Crustacea, providing an important staging point that will now facilitate the next stages of neuroendocrine research across the wider community.", "keywords": [ "Crustaceans", "Neuropeptides", "Crustacean Hyperglycemic Hormone (CHH)", "Transcriptomics", "Comparative genomics" ], "content": "Introduction\n\nCrustaceans and insects from the phylum Arthropoda have longstanding histories in peptide biology research, principally in areas related to the roles of peptide hormones in physiology and neuroendocrine signaling. Early discoveries have demonstrated that compounds in the crustacean nervous system were responsible for chromatophore control1–3. Four decades later, it was revealed that a compound known as the red pigment concentrating hormone functions as the first crustacean/invertebrate neuropeptide4. Since then, multiple studies have shed light on the highly pleiotropic functions of crustacean neuropeptides implicated in the regulation of a myriad of physiological processes such as light adaptation, molt inhibition, carbohydrate metabolism, reproduction and ion transport5–9.\n\nThe crustacean hyperglycemic hormone (CHH) represents a neuropeptide superfamily that is unique to arthropods6,10–12. This superfamily is made up of peptides containing ∼70 amino acids originally isolated from the X-organ-sinus-gland system of the decapod Carcinus maenas13. Given their high degree of structural similarities, and the conservation of six cysteine residues, the molt-inhibiting hormone (MIH) and gonad-inhibiting hormone (GIH) were considered as part of this family collectively known as CHH/MIH/GIH. To date, at least 150 CHH peptides have been isolated and characterized, mainly in decapods through comparative studies on endocrinology5–7,14–21. Although there are reports on CHH peptides in other crustacean taxa such as Armadillidium vulgare (Isopoda)22,23, Daphnia pulex (Cladocera)24 and Daphnia magna15, investigations beyond decapods have remained scant and the sequences of CHH/MIH/GIH genes in other crustacean taxa have remained elusive.\n\nHere, we took advantage of the growing number of high-throughput crustacean datasets on public repositories to perform transcriptome mining of the CHH/MIH/GIH superfamily. To this end, we looked at crustacean species from three Classes (Figure 1) and annotated CHH/MIH/GIH genes. This high confidence set of genes identified using our in silico framework provides an important basis for understanding neuropeptide biology underpinning physiological adaptations across diverse crustacean species.\n\nThe number of species within each taxon is denoted in parentheses.\n\n\nMethods\n\nWe retrieved complete transcriptome datasets for 112 crustacean species available at the time of manuscript preparation from the European Nucleotide Archive. Five non-crustacean arthropod proteomes were retrieved from Uniprot. A complete list of accessions used in this study is provided in Supplementary Table 1. We retrieved a list of query sequences used in subsequent homology searches from Uniprot and GenBank.\n\nTo identify CHH/MIH/GIH gene orthologs, we used multiple Basic Local Alignment Search Tool (BLAST)-based approaches such as BLASTp and tBLASTn with varying Blocks Substitution matrices based on a previously published workflow25. The BLAST results were filtered by e-value of < 10-6, best reciprocal BLAST hits against the GenBank non-redundant (nr) database and redundant contigs having at least 95% identity were collapsed using CD-HIT. We then utilized HMMER (version 3.1) employing hidden Markov models (HMM) profiles26 to scan for the presence of CHH Pfam domains27 on the best reciprocal nr BLAST hits to compile a final non-redundant set of crustacean CHH/MIH/GIH orthologs. Pfam annotations, associated e-values and fasta sequences are provided in Dataset 128 and Dataset 229.\n\nMultiple sequence alignments of CHH protein sequences were performed using MAFFT (version 7)30. Phylogenetic tree was built from the MAFFT alignment using RAxML WAG + G model to generate best-scoring maximum likelihood trees31. Geneious (version 7) was used to generate multiple sequence alignment images as well as graphical representations of the Newick tree32.\n\nWe have annotated CHH/MIH/GIH genes from 112 crustacean transcriptome datasets representing three Classes: Malacostraca (Amphipoda: 56 species, Decapoda: 14 species, Isopoda: 27 species, Euphausiacea: 2 species and Mysida: 1 species), Branchiopoda (3 species), and Copepoda (9 species) (Supplementary Table 1). We also looked at 5 non-crustacean species from Arthropoda: Insecta (3 species), Arachnida (1 species) and Chilopoda (1 species) (Supplementary Table 1). Using sequence and motif similarity based approaches, we have conservatively identified a total of 413 genes from these transcriptomes (Figure 2; Dataset 128 and Dataset 229).\n\nHeat maps denote the number of CHH/MIH/GIH genes identified from each crustacean species. CHH/MIH/GIH genes from five non-crustacean species within Arthropoda are also shown.\n\nMultiple sequence alignment analyses on representative CHH/MIH/GIH sequences revealed the presence of a conserved set of six cysteine residues (Figure 3), likely contributing to the formation of disulfide bonds33. Comparison of insect sequences from Drosophila melanogaster, Anopheles gambiae and Aedes aegypti demonstrated sequence identities of at least 46% (Supplementary Table 2). Within crustacean taxa, a range of sequence identities were observed: Branchiopoda (∼25% to 93%), Copepoda (∼12% to 30%) and Malacostraca (∼10% to 98%) (Supplementary Table 2). This is reflected in the phylogeny where CHH/MIH/GIH sequences from related individuals form distinct clusters (Figure 4). It was previously reported that multiple gene duplications of CHH family peptides occurred in the decapod lineage leading to a high degree of genetic polymorphism15, hence providing an explanation for our current observation. Two separate clusters of CHH genes exhibiting antagonistic patterns of expression were identified in the decapod Metapenaeus ensis, posited to represent an ancient gene duplication event34. Although it is not possible to pinpoint the genomic loci of CHH sequences identified from this study, it is likely that paralogous copies offer mechanisms for evolving new functions through functional divergence. CHH-like genes arising from duplication of the ancestral copy are subjected to reduced selective pressure and therefore may lose their hyperglycemic activity to adopt more specialized roles15. Further biochemical studies will be required to unravel the functions of the novel genes identified from this study.\n\nSix conserved cysteine residues are annotated within red boxes.\n\nThe tree was constructed using the maximum-likelihood method from an amino acid multiple sequence alignment. The node labels of each taxon are marked with distinctive colors denoted in the figure inset. Bootstrap support values (n=1000) are denoted as branch labels.\n\n\nConclusions\n\nWe have generated a high confidence list of CHH/MIH/GIH sequences from distantly related crustaceans. As a fundamental step in a broader endeavor this data is now available to the wider community to allow detail functional analyses pertinent to the next stages of neuropeptide research. Given the paucity of CHH sequences beyond decapod crustaceans, our analysis forms a promising basis for studies ranging from biochemistry to the evolution of this elusive superfamily.\n\n\nData availability\n\nData supporting the conclusions of this study are provided as Supplementary Material and Dataset 1 and Dataset 2.\n\nDataset 1: Fasta file for CHH/MIH/GIH sequences in crustaceans and other arthropods. 10.5256/f1000research.13732.d19119428\n\nDataset 2: List of Pfam annotated CHH/MIH/GIH genes and associated e-values in crustaceans and other arthropods. 10.5256/f1000research.13732.d19119529", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the EMBO Fellowship and the Human Frontier Science Program Fellowship to AGL.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Table 1: List of accession numbers for species used in this study.\n\nClick here to access the data.\n\nSupplementary Table 2: Protein distance matrix (% identity) constructed from a multiple sequence alignment of CHH/MIH/GIH sequences.\n\nClick here to access the data.\n\n\nReferences\n\nBrown FA: The Controlling Mechanism of Chromatophores in Palaemonetes. Proc Natl Acad Sci U S A. 1933; 19(3): 327–329. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkins EB: Color changes in crustaceans, especially in Palaemonetes. J Exp Zool Part A Ecol Genet Physiol. 1928; 50(1): 71–105. Publisher Full Text\n\nKleinholz LH: Crustacean eye-stalk hormone and retinal pigment migration. Biol Bull. 1936; 70(2): 159–184. Publisher Full Text\n\nFernlund P, Josefsson L: Crustacean color-change hormone: amino acid sequence and chemical synthesis. Science. 1972; 177(4044): 173–175. PubMed Abstract \| Publisher Full Text\n\nBöcking D, Dircksen H, Keller R: The crustacean neuropeptides of the CHH/MIH/GIH family: structures and biological activities. In Crustacean Nerv Syst. 2002; 84–97. Publisher Full Text\n\nChan SM, Gu PL, Chu KH, et al.: Crustacean neuropeptide genes of the CHH/MIH/GIH family: implications from molecular studies. Gen Comp Endocrinol. 2003; 134(3): 214–219. PubMed Abstract \| Publisher Full Text\n\nChristie AE, Stemmler EA, Dickinson PS: Crustacean neuropeptides. Cell Mol Life Sci. 2010; 67(24): 4135–4169. PubMed Abstract \| Publisher Full Text\n\nChristie AE, Cashman CR, Brennan HR, et al.: Identification of putative crustacean neuropeptides using in silico analyses of publicly accessible expressed sequence tags. Gen Comp Endocrinol. 2008; 156(2): 246–264. PubMed Abstract \| Publisher Full Text\n\nChristie AE, McCoole MD, Harmon SM, et al.: Genomic analyses of the Daphnia pulex peptidome. Gen Comp Endocrinol. 2011; 171(2): 131–150. PubMed Abstract \| Publisher Full Text\n\nBeltz BS: Crustacean neurohormones. Invertebr Endocrinol. 1988; 2: 235–258. Reference Source\n\nDe Kleijn DP, Van Herp F: Molecular biology of neurohormone precursors in the eyestalk of Crustacea. Comp Biochem Physiol B Biochem Mol Biol. 1995; 112(4): 573–579. 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PubMed Abstract \| Publisher Full Text\n\nVan Herp F: Molecular, cytological and physiological aspects of the crustacean hyperglycemic hormone family. Recent Adv Arthropod Endocrinol. 1998; 53–70. Reference Source\n\nGrève P, Sorokine O, Berges T, et al.: Isolation and amino acid sequence of a peptide with vitellogenesis inhibiting activity from the terrestrial isopod Armadillidium vulgare (Crustacea). Gen Comp Endocrinol. 1999; 115(3): 406–414. PubMed Abstract \| Publisher Full Text\n\nMartin G, Sorokine O, Van Dorsselaer A: Isolation and molecular characterization of a hyperglycemic neuropeptide from the sinus gland of the terrestrial isopod Armadillidium vulgare (Crustacea). Eur J Biochem. 1993; 211(3): 601–607. PubMed Abstract \| Publisher Full Text\n\nGard AL, Lenz PH, Shaw JR, et al.: Identification of putative peptide paracrines/hormones in the water flea Daphnia pulex (Crustacea; Branchiopoda; Cladocera) using transcriptomics and immunohistochemistry. Gen Comp Endocrinol. 2009; 160(3): 271–287. PubMed Abstract \| Publisher Full Text\n\nLai AG, Aboobaker AA: Comparative genomic analysis of innate immunity reveals novel and conserved components in crustacean food crop species. BMC Genomics. 2017; 18(1): 389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinn RD, Clements J, Eddy SR: HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011; 39(Web Server issue): W29–37. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBateman A, Coin L, Durbin R, et al.: The Pfam protein families database. Nucleic Acids Res. 2004; 32(Database issue): D138–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang WH, Lai AG: Dataset 1 in: Comparative genomic analysis of crustacean hyperglycemic hormone (CHH) neuropeptide genes across diverse crustacean species. F1000Research. 2018. Data Source\n\nChang WH, Lai AG: Dataset 2 in: Comparative genomic analysis of crustacean hyperglycemic hormone (CHH) neuropeptide genes across diverse crustacean species. F1000Research. 2018. Data Source\n\nKatoh K, Asimenos G, Toh H: Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol. 2009; 537: 39–64. PubMed Abstract \| Publisher Full Text\n\nStamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–3. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKearse M, Moir R, Wilson A, et al.: Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12): 1647–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChristie AE: Identification of the first neuropeptides from the Amphipoda (Arthropoda, Crustacea). Gen Comp Endocrinol. 2014; 206: 96–110. PubMed Abstract \| Publisher Full Text\n\nGu PL, Yu KL, Chan SM: Molecular characterization of an additional shrimp hyperglycemic hormone: cDNA cloning, gene organization, expression and biological assay of recombinant proteins. FEBS Lett. 2000; 472(1): 122–128. PubMed Abstract \| Publisher Full Text" }	[ { "id": "31165", "date": "26 Feb 2018", "name": "Abigail Elizur", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe current study is an interesting “in silico” approach that mine for CHH/MIH/GIH neuropeptide in a wide range of crustacean. Introduction is updated and concise. However, there are some minor points that will need to be addressed to provide clarity information for readers. Materials and methods is sound and appropriate, although I raise some particular suggestions to further expand the quality of the MS. Results and discussions are overall good. One major point that needs to be fixed is the phylogenetics tree, whereas the color code system does not correlate between legend and figure.\n\nIn addition, I have specific comments, (the fact that it does not appear as numbered pages and lines makes it a bit awkward, but see below):\n\nPage 2: …known as CHH/MIH/GIH- There are also Mandibular Inhibiting hormone (MOIH) and also ITP (Ion Transport Protein) to be part of the super family. See Ohira (2016)1.\n\n…from three Classes - Please elaborate on this, are these branchiopoda,. malacostraca and copepoda?\n\n(Figure 1) – How was Figure 1 generated ? Is this a phylogenetics based tree built on mitochondrial genome or it is just an illustration?\n\nGenebank nr database… What about TSA (Transcriptome Shotgun Assembly) database? As that will increase the chance that the algorithm will discover more CHH/MIH/GIH? Since you mention in the introduction \"Here, we took advantage of the growing number of high-throughput crustacean datasets on public repositories to perform transcriptome mining of the CHH/MIH/GIH superfamily.\" Adopting the TSA database will be better suited for the MS.\n\n…and redundant contigs… Why 95%?, let's assume that CHH prepropeptide can be roughly more than 120aa. For instance, two distinct CHH in Homarus americanus CHH-A (P19806) and CHH-B (Q25154) have 127/134 aa that are similar (~95%). Will they be collapsed with CD-HIT?\n\nPage 4 Within crustacean taxa… Similarity was pretty high in Branchiopoda and Malacostraca (93% & 98%), but significantly lower in Copepoda. Can you propose an explanation for that?\n\nIt was previously reported that… In light of this, Veenstra (2016)2 has some very intensive mining of CHH in multiple decapod species. Why not provide a section that compares between the number of CHH/MIH/GIH predicted from your dataset with the above study?\n\nFigure 4 - Color coding system is misleading, for instance Decapoda was assigned as teal in legends, but actually blue in the phylogenetics tree.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "30523", "date": "20 Apr 2018", "name": "Chi-Ying Lee", "expertise": [ "Reviewer Expertise Endocrinology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors collected and analyzed transcriptome datasets for 112 crustacean species (representing three Classes - Malacostraca, Branchiopoda, and Copepoda), as well as those for 5 non-crustacean (arthropod) species (Insecta, Arachnida and Chilopoda) (Supplementary Table 1; Fig. 1), retrieved from public repository.\n\nCollectively, 413 genes are annotated based on sequence and motif similarity as encoding peptides belonging to the so-called CHH/MIH/GIH family. The number of CHH/MIH/GIH genes identified from each crustacean species is shown as heat maps (Fig. 2). Protein sequence, annotation information, etc. are listed (Dataset 1 and 2). Between-species % identity is reported (Supplementary Table 2). Multiple sequence alignment on representative CHH/MIH/GIH sequences is presented in Fig. 3 to show the 6 conserved cysteine residues (Fig. 3). A phylogenetic tree is built based on the multiple sequence alignment data (Fig. 4).\n\nThe main drawback of this work is it only results in a 413-sequence inventory of CHH/MIH/GIH family peptides, but with little information (nucleotide length, amino acid sequence).\nThe authors did not describe how the tree presented in Fig. 1 was built with what sorts of data. If they took a constructed tree from other source, this should be cited. There are obviously other current interpretations of a phylogenetic relationship of Crustacea, in addition to the one shown in the manuscript.\nFor annotation information (Dataset 2), it should be more discerning in that peptide should be assigned as CHH, MIH/GIH, MOIH (mandibular organ-inhibiting hormone) or ITP (ion transport peptide), instead of only giving an inclusive description - Crustacean CHH/MIH/GIH neurohormone family.\nResults derived from further analysis of the sequence data (Figs, 2, 3, and 4) are not adequately discussed or lead to conclusions that have been previously established. For example, the only conclusion made with Fig. 3 is the 6 highly cysteine residues, a signature of the family already extensively described. Figure 4 is left without discussion. Moreover, it is not mentioned which type of peptide (CHH, MIH/HIV, or ITP) the different clusters highlighted in the tree belong to.\nPart of the discussion (left column, p. 4) starting with “It was previously reported that multiple gene duplications of CHH family peptides…..” is not particularly relevant to the data being discussed and is again making a point already extensively described and discussed. It should also be mentioned that genomic data are more adequate than transcriptomic data when discussing gene duplication and gene copy number (Fig. 2). Figure 2 is also left without discussion. In addition, data presented in this figure might be suffering from the criticism that the number of CHH/MIH/GIH genes assigned to each species is likely biased as the number will be absolutely influenced by the type of tissue used for sequencing (eyestalk ganglia, other tissue, or whole animal) and sequencing depth. Accordingly, the relevant information should be given in Supplementary Table 1 and comparison should only be made among species with the same sequenced tissue type (e.g., eyestalk ganglia) and comparable sequencing depth.\n\nOverall, while the methods employed are largely (but not entirely) appropriate for the intended analysis, the goal of the study is not clearly set and this work only produces a sequence inventory without novel finding or solid discussion. Additional analyses expected to yield novel finding should be added to the manuscript. For example, with a vast amount of sequence information that could be extracted from a set of 413 peptides from animals encompassing 3 Classes, would it not be possible to uncover some function-defining sequence characteristics or motif? This piece of information would be useful for functional analysis, which the authors thought should be an important aspect of the next stage of research.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "37870", "date": "08 Oct 2018", "name": "Jos H. M. Schippers", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript presented by Chang and Lai describes a collection of CHH neuropeptide genes in over 100 crustacean species.\nI read with interest this short report and could appreciate that a majority of neuropeptide studies have been limited to decapod crustacean species previously. In addition the presence of neuropeptide transcripts in other divergent crustaceans including those from basal lineages (copepods and branchiopods) are presented.\nLike in their previous work (Chang and Lai, 2018, BMC Genomics1), a broad sampling strategy was followed. Here the approach was across multiple crustacean lineages, which allows for drawing conclusions on the evolutionary trajectory of CHH genes and their potential functional role during life histories.\nAlthough the paper describes a single finding, it is a valuable asset, as it provides an inventory of a high-quality list of carefully curated CHH neuropeptide transcripts for future functional studies.\nThe paper is well-written and concise, and definitely fits with the scope and eligibility of the F1000Research guidelines.\n\nAs a minor comment: it would be good to describe how the tree in Figure 1 was made in the legend.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-100
https://f1000research.com/articles/4-163/v1	23 Jun 15	{ "type": "Research Article", "title": "Effects of conventional immunosuppressive therapy on functional and pathological features of CNS lupus in NZB/W mice", "authors": [ "Jessy Alexander", "Alexander Jacob", "Richard J. Quigg", "Alexander Jacob", "Richard J. Quigg" ], "abstract": "Neurological involvement is one of the most devastating complications of the disease, systemic lupus erythematosus (SLE). To understand the effect of the drugs, cyclophosphamide (CY) and prednisolone (PD) on CNS manifestations, the New Zealand Black/White (NZB/W) lupus mice, were given a cocktail of both drugs by intraperitoneal injections daily from 22 to 44 weeks of age. The treatment prolonged survival (10% of the treated 20 NZB/W mice died compared to 50% of the 30 NZB/W mice, with no mortality in the control NZW mice). Real-time PCR analysis showed a three- to fifteen-fold increase in the expression of GFAP, vimentin and syndecan4 in the cerebral cortex of 44 week NZB/W mice. These alterations were prevented by CY and PD treatment. Immunostaining revealed increased GFAP expression in NZB/W mice compared to congenic, nondiseased NZW mice, which was prevented by treatment. In addition, concomitant changes were observed in the expression of extracellular matrix proteins, collagen IV and fibronectin. To determine the impact of these alterations on the neurological manifestations of SLE, behavior was studied in these mice. The NZB/W mice were spontaneously less active in the open field and exhibited a decrease in distance traveled (58% of control, p<0.01) and ambulatory measurements (52% of control, p<0.01). They took more time (8.8+1.2min) to escape from the maze compared to the control NZW mice (2.6+0.8min). Even more striking was that the behavioral deficits were alleviated in these mice by CY and PD treatment. These results support the hypothesis that increased astrogliosis and altered extracellular matrix proteins may be two of the critical factors that mediate lupus brain disease.", "keywords": [ "Systemic lupus erythematosus", "central nervous system", "NZB/W mice", "behavior", "astrogliosis", "cyclophosphamide" ], "content": "Introduction\n\nSystemic lupus erythematosus (SLE) is an autoimmune disease in which one third of patients exhibit neuropsychiatric (NP) disturbances. Patients with NP-SLE have a variety of behavioral and cognitive impairments1–3. These are likely to track with pathological alterations occur in the brain, however subtle. A challenge in defining the pathogenesis of SLE is its complexity, involving affecting many systems and pathways.\n\nAnimal models have proven invaluable in our understanding of SLE4. Lupus mice mirror many of the findings seen clinically and they can be manipulated to help determine underlying pathophysiological mechanisms. Arguably the most accurate SLE model occurs in females of the F1 cross between New Zealand Black and New Zealand White mice (NZB/W)4,5. Among the finest examples of translational research occurred over a several decade period in work done at the National Institutes of Health by Alfred Steinberg, James Balow and colleagues; they first showed cyclophosphamide (CY) and methylprednisolone (PD) were efficacious in NZB/W mice6,7, followed by clinical studies in human SLE8 with this therapy remaining the “gold standard” by which all else is compared.\n\nNZB/W mice have an increase in anxiety behavior and decreased exploratory behavior, which is increased with advancing age, indicating these behaviors were related to the development of autoimmune disease2. In studies using the MRL/lpr lupus mouse model, these mice explored the open field less, spent more time at home-base, had impaired exploratory activity and defecated less in comparison to congenic MRL/+ controls1,3,9–12. Lupus mice have a progressive disease over time, including NP-SLE9, which does contrast to the periods of disease quiescence punctuated by intermittent flares seen in human SLE. Of course, the clinical disease is typically treated aggressively. Although the mice differ in this aspect from humans, they give us insights and a better understanding of the underlying mechanisms that lead to disease and enable the development of therapeutic strategies.\n\nSeveral alterations have been observed affecting the central nervous system (CNS) in lupus mice13. The cross-talk between the peripheral immune system and the CNS predicts that, particularly when there is an alteration of the blood-brain barrier (BBB)14, peripheral immune signals could result in glial cell activation. Activated astrocytes could secrete factors that promote neuronal survival, and could also initiate an inflammatory response, leading to neuronal death15, which could result in behavioral changes.\n\nThe CNS responds to injury by the process of gliosis which involves astrocytes, oligodendroglial precursors and meningeal cells. Astroglia are the most abundant glial cells in the CNS. They play a crucial role in maintaining normal brain physiology, integrity of the BBB16 and are a key component of the CNS response to injury and disease17–19. Astrocytes contain abundant intermediate filaments dispersed in the cell body and organized as thick bundles in astrocytic processes. Depending on the region of the brain, astrocytic intermediate filaments are either homopolymers of glial fibrillary acid protein (GFAP) or heteropolymers of GFAP and vimentin20,21. Reactive astrogliosis, in which astrocytes undergo hypertrophy or proliferation along with other histological and enzymatic changes, is a prominent feature in CNS inflammation22,23. Alterations in astrocytes could affect brain circuits that include interactions between neurons and astrocytes, thereby impacting behavior. Activation of astrocytes could lead to increased expression of the proteoglycan, syndecan, to provide a supporting environment for axons to regenerate at the site of brain injury24. Coupling of astrocytic and neuronal activities gives rise to membrane potential instability and oscillations25. The presence of autoantibodies to GFAP in serum correlated with NP manifestations in SLE patients26.\n\nAlthough there has been progress in our understanding of the immunology and phenotype of lupus brain disease, our current therapy is still imperfect. The continued use of non-specific and potent immunosuppressive agents like CY and PD is less than ideal. There remains a balance between drug toxicity and efficacy. Pulse doses of intravenous CY are often used for NP-SLE27. Treated patients show considerable clinical and electrophysiological improvement of cerebral function28. Children with severe neuropsychiatric lupus also showed a favorable response to this treatment29. In a similar manner, CY treatment alleviated symptoms in lupus mouse models including reduced leukocyte (CD45) infiltration in MRL/lpr mice and prevention of behavioral changes such as floating in the forced swim test30.\n\nHere we evaluated the effect of conventional CY/PD treatment on clinicopathological features of NP-SLE such as anxiety disorder and cognitive dysfunction. Our studies show that alterations in gliosis and extracellular matrix proteins tracked with behavioral changes in NZB/W mice, both of which were ameliorated by CY/PD treatment.\n\n\nMaterials and methods\n\nNew Zealand Black and New Zealand White mice were from Jackson Laboratories (Bar Harbor, ME) and crossed in-house to obtain NZB/W F1 mice. Mice were maintained in 12 h light and dark cycles (lights off at 6:00 p.m., lights on at 6:00 a.m.) and a temperature-controlled environment (21 ± 1°C). All studies were approved by the Animal Care and Use Committee at the University of Chicago.\n\nA total of 60 female mice were divided into three groups: 1) NZB/W mice treated from 22–44 weeks of age with CY/PD (each 3 mg/kg/d in saline) given once daily via intraperitoneal (i.p.) injection (n=20); 2) NZB/W mice treated identically, but without CY/PD (i.e., receiving saline vehicle only i.p.) (n=30); and, 3) NZW mice serving as controls (n=10).\n\nFollowing behavioral testing at 44 weeks of age, animals were euthanized. For this, animals were first anesthetized with inhalational isoflurane. Animals were documented to be unresponsive to pain, prior to proceeding. Cardiac puncture was performed with a 21 gauge needle followed by cervical dislocation to ensure euthanasia prior to brain tissue harvest. These procedures are consistent with the Panel on Euthanasia of the American Veterinary Medical Association (https://grants.nih.gov/grants/olaw/Euthanasia2007.pdf).\n\nTesting: All mice in a testing group were cage changed on the same day and no testing was performed until 1 day after a cage change. All behavioral testing was executed during the day, when the mice are normally active, with testing carried out in a behavioral testing room under normal light. To ensure the 5 min open field test was accurately measuring the activity of the mice, open field activity was measured over a 60 min period.\n\nPreliminary workup: A series of preliminary observations31 of general health, home cage behaviors and neurological reflexes (eye blink, response to tail pinch and righting reflex) were first conducted for each mouse to avoid spurious false positives. All mice tested were groomed (the appearance of its fur and whiskers is noted), and moved around the cage normally.\n\nOpen-field analysis: Mice were assessed using an open-field activity monitor (Med Associates Inc., St Albans, VT, USA) for a period of 60 min. The testing chamber was wiped clean with water and then with ethanol between each test subject. The chambers were kept in a room used only for behavioral studies so that no movement or sound disturbs the behavior of the mice. Each subject was placed in the center of an open field apparatus. Measurements (calculated total distance traversed, number of movements, horizontal activity, vertical active and resting time and the beam–break counts for stereotyped behaviors) were recorded by accompanying software (Med Associates Inc, Activity Monitor, version 5.1) and calculated using Excel software (Microsoft, 2007). Ambulatory count and episodes were also recorded. Ambulatory count was defined as the number of beam breaks while the mouse is ambulating, while ambulatory episodes are the number of times the mouse begins ambulating (from a resting position). Data were collected over a 60-min period.\n\nMaze: A circular maze having three chambers of 4, 8 and 12 inches was constructed. The mice were placed in the middle chamber. The time taken by the mice to escape from the maze was recorded.\n\nReal-time quantitative (q) RT-PCR was performed on RNA from cerebral cortices dissected from NZW, NZB/W and CY/PD-treated NZB/W mice (n=10 each). RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and cleaned (RNeasy Mini Kit, Qiagen, Valencia, CA, USA). Total RNA (1 µg) from each sample was reverse transcribed with random hexamer primers using M-MuLV reverse transcriptase (Life Technologies). Ten ng of cDNA and gene-specific primers were added to SYBR Green PCR Master Mix (SYBR Green I Dye, AmpliTaq DNA polymerase, dNTPs with dUTP and optimal buffer components; Applied Biosystems, Foster City, CA, USA) and subjected to PCR amplification (one cycle at 50°C for 2 min, one cycle at 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min) in a TaqMan 5700 Sequence Detection System (Applied Biosystems). For each transcript, real-time PCR was conducted three times in duplicate using each of the RNA samples. The amplified transcripts were quantified with the comparative CT method using GAPDH RNA as the control (http://docs.appliedbiosystems.com/pebiodocs/04303859.pdf). The primers were designed using the Primer Express software (Applied Biosystems) based on the GenBank accession numbers; the sequences (5'−3') are as follows:\n\nSections of 6μm thickness were incubated with a blocking solution for 30 min, then with the primary antibodies overnight at 4°C. After washing in PBS, the sections were incubated in secondary fluorescence-conjugated antibodies (1:200) for 1 h at room temperature, washed in PBS and cover slips placed on them. Polyclonal antibodies used and their respective dilutions in PBS were: FITC-conjugated antibodies to mouse C3 (1:200, Cappel, 55500); rabbit anti-GFAP (1:200 Dako, CA, USA, Z0334), rabbit anti-collagen IV (1:60, Sigma, SAB4500369), anti-fibronectin (1:60, Sigma, F3648) followed by goat anti-rabbit-Alexa Fluor 594 (A-21207, 1:100), and 488 (A-11034, 1:100) respectively (Molecular Probes). Negative controls were generated by omitting either primary or secondary antibodies. Images were acquired with a Zeiss microscope. The sections were photographed maintaining the exposure time constant (Axiocam version 3.1; Zeiss).\n\nStatistical analyses were performed using Minitab software (v. 17.1, State College, PA, USA). The number of control NZB/W mice included an estimated 40–60% mortality by 44 weeks and the positive effects of CY/PD treatment6,7, with the resultant informative censoring of data32. Power analyses were done for one-way ANOVA with three levels, assuming α = 0.05 and β = 0.2 (power = 0.8). Sample sizes of 10 and 20 were sufficient to identify changes of 1.0 and 1.5 times the SD.\n\nThe data from each mouse in the study are presented graphically; group means are also depicted. Statistical significance was determined by one-way ANOVA; a P-value < 0.05 was used to reject the null hypothesis of no differences among the level means. Tukey’s method was then used for pairwise comparisons, with resultant P-values presented in the appropriate section.\n\n\nResults\n\nThe study was begun with 50 female NZB/W as well as 10 NZW control mice. At 22 weeks of age, 30 of NZB/W mice began treatment with CY and PD, which was administered during the full evolution of autoimmune disease, until 44 weeks when they were sacrificed. At 30 weeks the first untreated NZB/W mouse was found dead. Thereafter, there was progressive mortality, such that by the end of the study at 44 weeks only 15 NZB/W mice (50% of the starting number) remained. There were only two dead among the NZB/W that were treated (10% of the starting number) and no mortalities among the NZW controls (Figure 1).\n\nA series of preliminary observations were first conducted prior to proceeding with testing31. These included assessments of general health, home cage behavior and neurological reflexes (eye blink, response to tail pinch, and righting reflex). All mice tested had appropriate grooming of fur and whiskers, and moved around the cage normally. The NZB/W mice had tail pinch reflex and their body weight remained the same.\n\nOpen field behavior in rodents is considered to be a fundamental index of their general behavior and inability to escape from a maze is considered as a sign of anxiety. As shown in Figure 2A, on average, control NZW mice traversed over 3,000 cm over 60 min. Untreated NZB/W mice had considerable hypoactivity compared to NZW control mice, which was largely prevented with CY/PD treatment (Figure 2A). Similar patterns were observed in ambulatory and stereotypic (i.e., animal rearing/vertical movement). The number of jumps did not differ between the groups (Supplementary Dataset 1). Control NZW mice escaped from the maze within 1–3 min, while untreated NZB/W mice demonstrated anxious/timid behavior by staying close to the walls of the maze, with all requiring more than 6 min to escape, including two animals that remained within the maze at 10 min (Figure 2B). Treatment with CY/PD reduced but did not fully reverse this phenomenon (Figure 2B).\n\nThe ambulatory count, stereotypic count and average distance traveled by 44 week old NZW, and NZB/W mice treated CY/PD (NZB/W-T) or saline control were recorded for 60 min. Ambulatory episodes, vertical rearings and number of jumps were recorded for 60 min. The NZB/W mice covered significantly less distance and had significantly less number of vertical counts compared to their CY/PD treated counterparts. The NZB/W mice took longer to escape from the maze compared to the NZW controls, staying closer to the walls of the maze. Mice treated with CY/PD depicted less anxiety and escaped from the maze significantly faster than the untreated lupus mice. In all four measurements, the means were different in the three groups by ANOVA. P < 0.05 vs. NZW and NZB/W-T. +P < 0.05 vs. NZW and untreated NZB/W.\n\nUpregulation of astrocytic intermediate filaments is a crucial step in astrocyte activation or astrocytosis. Therefore, we performed qPCR on cDNA derived from the brains of the mice in this study. As shown in Figure 3, the mRNA for GFAP, vimentin and syndecan 4 were all increased in NZB/W mouse brains. Treatment with CY/PD from 22 to 44 weeks of age almost completely prevented this upregulated expression of these genes (Figure 3).\n\nRNA of mouse brains was reverse transcribed to cDNA and then subjected to real-time qPCR with gene-specific primers as described in Methods. Data are presented as gene expression relative to GAPDH. Each point represents data from NZW mice and NZB/W mice treated with either saline or CY and PD from 22 to 44 weeks of age. In all three measurements, the means were different in the three groups by ANOVA. P < 0.05 vs. NZW and NZB/W-T.\n\nThe upregulated expression of GFAP mRNA in brains of NZB/W mice suggested the presence of astrogliosis (reactive astrocytosis). We therefore examined GFAP protein expression by IF microscopy. In NZW mice, there was low level expression of GFAP in the cortex (Figure 4A). In NZB/W mice, many GFAP-positive activated glia were observed in the cortex (Figure 4B), as well as the hippocampus (not shown). The number of GFAP-positive glia was reduced qualitatively by CY/PD treatment which tended to match the changes observed at the mRNA level for this protein.\n\nRepresentative sections from NZW, untreated NZB/W and CY/PD-treated NZB/W brains were stained for GFAP. Inset, is an astrocyte at 60x under oil. Note the more intense GFAP staining, thicker, shorter processes and swollen soma of astrocytes in the NZB/W sections compared with the controls and treated mice. CY/PD treatment significantly reduced the number of reactive GFAP-expressing astroglia in these brains.\n\nSince syndecan 4 associates with fibronectin and collagen IV, we determined their expression in the brains of NZB/W mice and it followed the same pattern of expression as the others, with and without treatment. Fibronectin (Figure 5A) and collagen IV (Figure 5B) were localized around the microvasculature. CY/PD treatment prevented the increase in extracellular matrix protein expression in lupus brains.\n\nAccumulation of fibronectin (A) and collagen IV (B) occurs in NZB/W lupus mouse brains which is reduced by CY/PD treatment. Representative cryosections were immunostained with rabbit anti-mouse fibronectin and detected using Alexa 594 labeled anti-rabbit antibody (A) and rabbit anti-mouse collagen and detected using Alexa 488 labeled anti-rabbit antibody (B). Fibronectin and collagen IV was localized mainly around the microvasculature. RNA of mouse brains was reverse transcribed to cDNA and then subjected to real-time qPCR with primers for fibronectin and collagen IV as described in Methods. Data are presented as gene expression relative to GAPDH. Each point represents data from NZW mice and NZB/W mice treated with either saline or CY and PD from 22 to 44 weeks of age. CY and PD treatment reduced the expression of these extracellular matrix molecules in these brains to levels observed in control NZW brains. In both measurements, the means were different in the three groups by ANOVA. *P < 0.05 vs. NZW and NZB/W-T.\n\n\nDiscussion\n\nSLE is a complex, autoimmune disease, and considerable work has been done in the field. Yet, the mechanism underlying the CNS pathology, with its tight regulation and specialized microenvironment, is not completely understood. It is a complex, systemic disease that might be mediated by the synergistic action of many factors. As expected in the setting of an autoimmune disease, IgG deposits were significantly increased in NZB/W lupus mice compared to the NZW controls, which was prevented by CY and PD treatment. Using the microarray technique which gives insight into global mRNA changes in brain, we observed increased expression of GFAP, vimentin and syndecan 4 genes in the cortex, which was prevented by CY and PD treatment.\n\nAstrocytes play a major role in regulating immune responses within the CNS and express major histocompatibility complex molecules required for antigen presenting cellular activity33. During neurodegeneration, astrocytes are activated and release both proinflammatory cytokines and chemokines34. Our results give insight into the potential contributions of intrinsic astroglial cellular hyper-responsiveness in the development of NP-SLE. Recent studies have shown that GFAP levels in SLE patients with CNS manifestations were 3-fold higher than those patients without CNS involvement, which was prevented by CY treatment. Furthermore, the GFAP level in these patients significantly correlated with MRI abnormalities35. A significant positive correlation was observed between anti-GFAP serum antibodies and NP manifestations26.\n\nAlthough we cannot exclude a possible relation between health deterioration and the behavioral abnormalities, we believe these studies are important as many of the changes were also observed in preclinical mice of 6 weeks of age. The NZB/W mice display more anxiety behavior, less activity, and less exploratory behavior than non-autoimmune female NZW mice, prior to and during disease emergence at 6 and 12 weeks of age, respectively2,36. The behavioral alterations observed prior to clinical disease suggest the participation of a genetic component to these differences. Concomitant with our findings of increased expression of astrogliosis and alteration in extracellular matrix proteins, the NZB/W mice showed significantly less activity than the NZW mice, based on the open-field behavior. Furthermore, they also demonstrated increased anxiety. These changes in behavior were prevented by treatment with CY/PD. These findings were similar to the alleviation of neuropsychiatric behavior observed in SLE patients treated with CY/PD28.\n\nFinally, this is the first attempt to understand the effect of CY/PD treatment in experimental NP-SLE, in a comprehensive manner. The treatment significantly alleviated both molecular and behavioral alterations in these mice, similar to humans. Future investigations elucidating the precise signaling mechanisms by which astrocyte activation occurs, prevent regeneration of neurons and contribute to the pathology observed in brain, in SLE, will reveal a critical location for therapeutic intervention in NP-SLE. In addition, GFAP in the cerebrospinal fluid could be used as indicators of brain damage and should be used as a follow-up tool in these patients.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw data for Figure 2, 10.5256/f1000research.6568.d4971437\n\nF1000Research: Dataset 2. Raw data for Supplementary Figure S1, 10.5256/f1000research.6568.d4971738\n\nF1000Research: Dataset 3. Raw data for Figure 3, 10.5256/f1000research.6568.d4971539\n\nF1000Research: Dataset 4. Raw data for Figure 5, 10.5256/f1000research.6568.d4971640\n\nF1000Research: Dataset 5. Behavioural data, 10.5256/f1000research.6568.d4971841", "appendix": "Author contributions\n\n\n\nJJA and AJ performed the studies. JJA and RJQ planned and designed the experiments and analyzed data. JJA, AJ, and RJQ wrote the manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by National Institutes of Health grants R01DK041873 and R01DK055357, and by the Arthur M. Morris Endowed Chair to RJQ.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nSakic B, Hanna SE, Millward JM: Behavioral heterogeneity in an animal model of neuropsychiatric lupus. Biol Psychiatry. 2005; 57(6): 679–687. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSchrott LM, Crnic LS: Anxiety behavior, exploratory behavior, and activity in NZB × NZW F1 hybrid mice: role of genotype and autoimmune disease progression. Brain Behav Immun. 1996; 10(3): 260–274. PubMed Abstract \| Publisher Full Text\n\nSzechtman H, Sakić B, Denburg JA: Behaviour of MRL mice: an animal model of disturbed behaviour in systemic autoimmune disease. Lupus. 1997; 6(3): 223–229. PubMed Abstract \| Publisher Full Text\n\nTheofilopoulos AN, Dixon FJ: Murine models of systemic lupus erythematosus. Adv Immunol. 1985; 37: 269–390. 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PubMed Abstract \| Publisher Full Text\n\nStojanovich L, Stojanovich R, Kostich V, et al.: Neuropsychiatric lupus favourable response to low dose i.v. cyclophosphamide and prednisolone (pilot study). Lupus. 2003; 12(1): 3–7. PubMed Abstract \| Publisher Full Text\n\nBaca V, Lavalle C, García R, et al.: Favorable response to intravenous methylprednisolone and cyclophosphamide in children with severe neuropsychiatric lupus. J Rheumatol. 1999; 26(2): 432–439. PubMed Abstract\n\nFarrell M, Sakić B, Szechtman H, et al.: Effect of cyclophosphamide on leukocytic infiltration in the brain of MRL/lpr mice. Lupus. 1997; 6(3): 268–274. PubMed Abstract \| Publisher Full Text\n\nCrawley JN, Paylor R: A proposed test battery and constellations of specific behavioral paradigms to investigate the behavioral phenotypes of transgenic and knockout mice. Horm Behav. 1997; 31(3): 197–211. PubMed Abstract \| Publisher Full Text\n\nSchluchter MD: Methods for the analysis of informatively censored longitudinal data. Stat Med. 1992; 11(14–15): 1861–1870. PubMed Abstract\n\nConstantinescu CS, Tani M, Ransohoff RM, et al.: Astrocytes as antigen-presenting cells: expression of IL-12/IL-23. J Neurochem. 2005; 95(2): 331–340. PubMed Abstract \| Publisher Full Text\n\nDong Y, Benveniste EN: Immune function of astrocytes. Glia. 2001; 36(2): 180–190. PubMed Abstract \| Publisher Full Text\n\nTrysberg E, Nylen K, Rosengren LE, et al.: Neuronal and astrocytic damage in systemic lupus erythematosus patients with central nervous system involvement. Arthritis Rheum. 2003; 48(10): 2881–2887. PubMed Abstract \| Publisher Full Text\n\nSchrott LM, Crnic LS: Increased anxiety behaviors in autoimmune mice. Behav Neurosci. 1996; 110(3): 492–502. PubMed Abstract \| Publisher Full Text\n\nAlexander J, Jacob A, Quigg RJ: Dataset 1 in: Effects of conventional immunosuppressive therapy on functional and pathological features of CNS lupus in NZB/W mice. F1000Research. 2015. Data Source\n\nAlexander J, Jacob A, Quigg RJ: Dataset 2 in: Effects of conventional immunosuppressive therapy on functional and pathological features of CNS lupus in NZB/W mice. F1000Research. 2015. Data Source\n\nAlexander J, Jacob A, Quigg RJ: Dataset 3 in: Effects of conventional immunosuppressive therapy on functional and pathological features of CNS lupus in NZB/W mice. F1000Research. 2015. Data Source\n\nAlexander J, Jacob A, Quigg RJ: Dataset 4 in: Effects of conventional immunosuppressive therapy on functional and pathological features of CNS lupus in NZB/W mice. F1000Research. 2015. Data Source\n\nAlexander J, Jacob A, Quigg RJ: Dataset 5 in: Effects of conventional immunosuppressive therapy on functional and pathological features of CNS lupus in NZB/W mice. F1000Research. 2015. Data Source" }	[ { "id": "27861", "date": "01 Dec 2017", "name": "Trine N Jørgensen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-powered and inclusive study, providing evidence that treatment with cyclophosphamide and prednisolone effectively limits signs of CNS lupus in a well-known spontaneous mouse model of lupus; the NZBxNZWF1 model. Despite a few missing words and misspellings, the manuscript is well-written and the conclusions are supported by the data.\nMinor comments:\nIn discussion it is claimed that \"the microarray technique..\" was used for determining relevant gene transcript levels in brain tissue, however in the methods section a real-time RT-PCR analysis is described. Please comment and correct accordingly.\nPlease read the manuscript, in particular the introduction, for complete sentences and rewrite as needed.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3343", "date": "22 Jan 2018", "name": "Jessy Alexander", "role": "Author Response", "response": "We thank the reviewer for the positive response.Response: The transcript levels were determined by qRT-PCR and the discussion has been corrected accordingly.Response: The manuscript has been read thoroughly and corrections made accordingly." } ] }, { "id": "28736", "date": "06 Dec 2017", "name": "Christopher Reilly", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is straight forward written showing the effects of immunosuppressive therapy on animal behaviour in mice with lupus. A couple questions remain. Is there a dose effect of drug on brain function? How much of the compounds actually get across the BBB to effect the cells. Finally a more detail analysis of the sections of the brain that were analysed would also make the manuscript better.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3344", "date": "22 Jan 2018", "name": "Jessy Alexander", "role": "Author Response", "response": "We thank the reviewer for constructive criticsms.Response: When studying pharmacological agents introduced into circulation and the brain, an important aspect is transport across the BBB. Both cyclophosphamide and prednisolone cross the BBB. The concentration of how much crosses the BBB and is there a dose effect is a kinetic study to be addressed in the future. Our current study shows that when the agents were administered as given to patients, they had a beneficial effect similar to that observed in patients strengthening the translational viability of the model and the studies. The sections of the brain used for analysis in the present study were the cortical regions of the brain." } ] } ]	1	https://f1000research.com/articles/4-163
https://f1000research.com/articles/7-93/v1	22 Jan 18	{ "type": "Research Article", "title": "Cell-specific cis-natural antisense transcripts (cis-NATs) in the sperm and the pollen vegetative cells of Arabidopsis thaliana", "authors": [ "Peng Qin", "Ann E. Loraine", "Sheila McCormick", "Peng Qin", "Ann E. Loraine" ], "abstract": "Background: cis-NATs (cis-natural antisense transcripts) are transcribed from opposite strands of adjacent genes and have been shown to regulate gene expression by generating small RNAs from the overlapping region. cis-NATs are important for plant development and resistance to pathogens and stress. Several genome-wide investigations identified a number of cis-NAT pairs, but these investigations predicted cis-NATS using expression data from bulk samples that included lots of cell types. Some cis-NAT pairs identified from those investigations might not be functional, because both transcripts of cis-NAT pairs need to be co-expressed in the same cell. Pollen only contains two cell types, two sperm and one vegetative cell, which makes cell-specific investigation of cis-NATs possible. Methods: We investigated potential protein-coding cis-NATs in pollen and sperm using pollen RNA-seq data and TAIR10 gene models using the Integrated Genome Browser. We then used sperm microarray data and sRNAs in sperm and pollen to determine possibly functional cis-NATs in the sperm or vegetative cell, respectively. Results: We identified 1471 potential protein-coding cis-NAT pairs, including 131 novel pairs that were not present in TAIR10 gene models. In pollen, 872 possibly functional pairs were identified. 72 and 56 pairs were potentially functional in sperm and vegetative cells, respectively. sRNAs were detected at 794 genes, belonging to 739 pairs. Conclusion: These potential candidates in sperm and the vegetative cell are tools for understanding gene expression mechanisms in pollen.", "keywords": [ "Pollen", "Sperm", "Cis-Natural antisense transcript", "Arabidopsis" ], "content": "Introduction\n\nNatural antisense transcripts (NATs) are endogenous transcripts that contain sequences complementary to each other. NATs have been shown to regulate gene expression by generating small RNAs from the overlapping region (Zhang et al., 2013). NATs are classified into two subgroups according to the site of their biogenesis: trans-NATs and cis-NATs. Trans-NATs are transcribed from different genomic loci, whereas cis-NATs are transcribed from opposite strands of adjacent genes (Jin et al., 2008). Based on the relative orientation and overlap degree of two transcripts, cis-NATs can be categorized into three types: head-to-head (5′ to 5′), tail-to-tail (3′ to 3′) and fully overlapping (Jin et al., 2008). cis-NATs are widely present in plants, animals and fungi (Zhang et al., 2013). In plants, cis-NATs are important for pathogen resistance (Katiyar-Agarwal et al., 2006), stress tolerance (Borsani et al., 2005), successful fertilization (Ron et al., 2010), and phosphate homeostasis and plant fitness (Jabnoune et al., 2013).\n\nSeveral genome-wide investigations identified potential cis-NATs in plants, ranging from 1057 to 1710 pairs in Arabidopsis (Henz et al., 2007; Jin et al., 2008; Wang et al., 2005; Zhan & Lukens 2013), and 3819 pairs in rice (Lu et al., 2012). However, all the expression data used in these cis-NAT investigations were from bulk samples such as seedlings, leaves or inflorescences, which include many cell types. For potential cis-NATs to be functionally relevant, the reverse and complementary transcripts must be co-expressed in the same cell. Some potential cis-NATs identified in those investigations might be expressed in different cells and, thus, the presence of overlapping transcripts in the same cell is not known. Moreover, the regions predicted to overlap in previous investigations were based on available gene model annotations, which might not fully represent potential overlaps, due to alternative splicing at different developmental stages or, to more extensive 5’ or 3’ UTRs than are annotated.\n\nA pollen grain contains only two types of cell, one vegetative cell and two sperm cells, and thus can be used to investigate cell-specific cis-NAT pairs. Pollen RNA-seq data (Loraine et al., 2013) provides accurate transcript lengths, helpful for precisely identifying overlapping regions of two adjacent genes. Sperm microarray data (Borges et al., 2008) is helpful for defining whether two adjacent genes are expressed in the same cell. Moreover, a small RNA database for pollen and sperm (Slotkin et al., 2009) can be used to determine if small RNAs were detected from any overlapping regions.\n\nIn this study, we investigated potential cis-NATs in Arabidopsis sperm and vegetative cells using pollen RNA-seq data, sperm microarray data and sRNA data in pollen and sperm. In total, we identified 1471 cis-NAT pairs, including 131 novel pairs, with 72 and 56 pairs being potentially functional cis-NATs in sperm and vegetative cells respectively. These cis-NATs are tools for understanding gene regulation mechanisms in sperm and vegetative cells.\n\n\nMethods\n\nWe investigated potential cis-NATs from protein-coding genes in pollen and sperm using pollen RNA-seq data (Borges et al., 2008; Loraine et al., 2013) and TAIR10 gene models (TAIR10 gene annotation data available here) using the Integrated Genome Browser, available from http://www.bioviz.org (Freese et al., 2016). We loaded TAIR10 gene models s and pollen RNA-seq into IGB, then manually scanned for cis-NATs in each chromosome, based on the following parameters: 1) the orientation of two adjacent genes in TAIR10 was reverse and complementary; 2) the length of transcripts mapping to the overlap of two adjacent genes was larger than 21nt, because the size of sRNA generated by cis-NATs is normally larger than 21 nt; and 3) both adjacent genes encoded proteins. The expression and sRNA data of each cis-NAT was merged with the cis-NAT data in Excel to generate sheet 1 of Table S1. Different categories of cis-NATs in sheets 2–8 of Table S1 were obtained based on the cis-NAT data in sheet 1 of Table S1.\n\n\nResults\n\nWe identified 1471 potential cis-NAT pairs, comprising 1373 pairs whose transcripts were complementary at their 3’ ends, and 98 pairs whose transcripts were complementary at their 5’ ends (Figure 1A, sheet 1 and 2 of Table S1). Among these 1471, in 37 pairs one transcript was completely internal to the other, 100 pairs comprised 50 sets that had three overlapping genes (sheet 1 of Table S1), and 131 pairs (8.9%) were not apparent using the TAIR 10 gene models, but were detected in the pollen RNA-seq data (Figure 1B and G, sheet 1 of Table S1).\n\nOne criterion for functional cis-NATs is that the two adjacent genes of a cis-NAT pair are expressed. To identify potentially functional cis-NATs in pollen, we analyzed the expression level of the 1471 gene pairs, defining genes with reads per million (RPM) ≥1 as expressed. There were 599 pairs for which the RPM of two adjacent genes was lower than 1 (sheet 3 of Table S1), suggesting that those 599 pairs might not produce relevant cis-NATs in pollen. Most sperm-specific genes, such as GEX1, GEX2 (Rutley & Twell, 2015), and Kokopelli (Ron et al., 2010), are detectable in pollen RNA-seq data, but sperm-specific genes with lower expression levels, such as ARI14, might not be detected, as the proportion of RNA from the vegetative cell is much larger than that from the sperm cells. So potential cis-NATs with one expressed gene in pollen RNA-seq data might still be functional in pollen. Based on this, there were 872 possibly functional cis-NATs pairs, for which the RPM of either or both adjacent genes was ≥1 (Figure 1C, sheet 4 of Table S1), of which 62 pairs did not overlap in TAIR10 gene models. Note that we did not detect a cis-NAT pair between Kokopelli and ARI14 (Ron et al., 2010), because the TAIR 10 gene model does not show them overlapping, and ARI14 expression is low in wild type. Thus it is possible that other cis-NATs might similarly not be included in sheet 4 of Table S1 (see below).\n\n(A) Overview of potential cis-NATs with different overlapping types. (B) Overview of potential cis-NATs present or non-present in TAIR 10 gene models. (C) Overview of potential cis-NATs based on reads per million (RPM) from pollen RNA-seq data. (D) Overview of potential cis-NATs possibly functional in sperm cells (SC). (E) Overview of potential cis-NATs possibly functional in the vegetative cell (VC). (F) Overview of potential cis-NATs with sRNAs in vegetative and sperm cells.(G) Potential cis-NATs not present in TAIR10 gene models, but detected in pollen RNA-seq data.\n\nAnother criterion for functional cis-NATs is that both adjacent genes are expressed in the same cell. In order to accurately identify potentially functional cis-NATs in pollen, we investigated the gene expression level of 872 pairs in sperm cells. The microarray signal calls “Present (P) or Absent (A)” (Borges et al., 2008) were used to categorize genes as expressed or not expressed, respectively. We identified 72 pairs for which both adjacent genes were expressed in sperm, supporting the likelihood that these 72 cis-NATs pairs exist in sperm (sheet 5 of Table S1). There were an additional 271 pairs for which only one of the adjacent genes was expressed in sperm (Figure 1D and sheet 6 of Table S1), some of which might pertain to the Kokopelli/ARI14 example. To test if these pairs might function in the vegetative cell, we defined the gene as expressed in the vegetative cell if either 1): both pollen and sperm signals were called “P”, and the ratio of the pollen to sperm signal was > 3; or 2): the pollen signal was “P” and sperm signal was “A”. This exercise yielded 56 pairs for which both adjacent genes were expressed in the vegetative cell (sheet 7 of Table S1), and 145 pairs for which only one was expressed in the vegetative cell (Figure 1E and sheet 8 of Table S1). Another hallmark of functional cis-NATs is that there are small RNAs generated from the overlapping region. We therefore investigated the sRNAs of pollen and sperm (Slotkin et al., 2009) for these potentially functional cis-NAT pairs. sRNAs were detected at 794 genes, belonging to 739 pairs (Figure 1F and sheet 1 of Table S1). Of these, 35 cis-NATs pairs had sRNAs from the overlapping region.\n\n\nDiscussion\n\ncis-NATs are widely present in plants, and play an important role in regulating gene expression. However, in plants precise identification of cis-NATs at a cell-specific level to support whether cis-NATs might be functional is lacking. One possible reason was that it is difficult to get specific cell types for RNA-seq. As pollen grains contain only two types of cell, they are an excellent model to investigate cell-specific cis-NATs.\n\nThe vegetative cell forms a pollen tube that transports two sperm cells into the ovule for fertilization. Successful fertilization needs proper gene regulation in both the vegetative and sperm cells (Ron et al., 2010). The precise identification of cis-NATs in pollen is a tool for understanding the molecular mechanism of pollen tube growth and fertilization. The 131 novel potential cis-NATs in pollen, and the 72 and 56 potentially functional cis-NATs in sperm and the vegetative cell, respectively, provide candidates toward further uncovering the regulatory mechanisms of gene expression in the sperm and vegetative cells.\n\n\nConclusion\n\nWe identified 1471 potential cis-NAT pairs, including 131 pairs only detected in the pollen RNA-seq data. There were 872 pairs expressed in the same cell and thus possibly functional in pollen, while 72 and 56 pairs were potentially functional in sperm and vegetative cell, respectively.\n\n\nData availability\n\nArabidopsis pollen RNA-seq alignments data were loaded into Integrated Genome Browser from the IGB Quickload site\n\nUnprocessed sequence data are available from the Sequence Read Archive under accession SRP022162 (Loraine et al., 2013).\n\nThe pollen and sperm microarray data was from (Borges et al., 2008) (available at https://bmcplantbiol.biomedcentral.com/articles/10.1186/1471-2229-9-87)\n\nsRNAs in Arabidopsis pollen and sperm were downloaded from https://mpss.danforthcenter.org/dbs/index.php?SITE=at_sRNA&lib_type=sRNA&lib_id=487 and https://mpss.danforthcenter.org/dbs/index.php?SITE=at_sRNA&lib_type=sRNA&lib_id=489, respectively (Slotkin et al., 2009).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by United States Department of Agriculture Current Research Information System (5335- 21000-030-00D) funding to S.M.\n\n\nAcknowledgements\n\nWe thank Dylan Ting and Andrew Shieh for helping investigate cis-NATs.\n\n\nSupplemental material\n\nTable S1. Investigation of cis-NATs in pollen and sperm cells of Arabidopsis.\n\nClick here to access the data.\n\nSheet 1) List of all potential cis-NATs identified using TAIR 10 gene models and pollen RNA-seq data; Sheet 2) List of potential cis-NAT pairs with three overlapping genes; Sheet 3) List of potential cis-NATs pairs for which the RPM of both adjacent genes is < 1; Sheet 4) List of potential cis-NATs pairs for which the RPM of either or both adjacent genes is ≥ 1; Sheet 5) List of potential cis-NAT pairs for which both adjacent genes are expressed in sperm cells; Sheet 6) List of potential cis-NAT pairs for which only one gene is expressed in sperm cells; Sheet 7) List of potential cis-NAT pairs for which both adjacent genes are expressed in the vegetative cell; Sheet 8) List of potential cis-NAT pairs for which only one gene is expressed in the vegetative cell.\n\n\nReferences\n\nBorges F, Gomes G, Gardner R, et al.: Comparative transcriptomics of Arabidopsis sperm cells. Plant Physiol. 2008; 148(2): 1168–1181. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBorsani O, Zhu J, Verslues PE, et al.: Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell. 2005; 123(7): 1279–1291. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFreese NH, Norris DC, Loraine AE: Integrated genome browser: visual analytics platform for genomics. Bioinformatics. 2016; 32(14): 2089–2095. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHenz SR, Cumbie JS, Kasschau KD, et al.: Distinct expression patterns of natural antisense transcripts in Arabidopsis. Plant Physiol. 2007; 144(3): 1247–1255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJabnoune M, Secco D, Lecampion C, et al.: A rice cis-natural antisense RNA acts as a translational enhancer for its cognate mRNA and contributes to phosphate homeostasis and plant fitness. Plant Cell. 2013; 25(10): 4166–4182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJin H, Vacic V, Girke T, et al.: Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis. BMC Mol Biol. 2008; 9: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatiyar-Agarwal S, Morgan R, Dahlbeck D, et al.: A pathogen-inducible endogenous siRNA in plant immunity. Proc Natl Acad Sci U S A. 2006; 103(47): 18002–18007. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoraine AE, McCormick S, Estrada A, et al.: RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing. Plant Physiol. 2013; 162(2): 1092–1109. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu T, Zhu C, Lu G, et al.: Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice. BMC Genomics. 2012; 13: 721. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRon M, Alandete-Saez M, Eshed Williams L, et al.: Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis. Genes Dev. 2010; 24(10): 1010–1021. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRutley N, Twell D: A decade of pollen transcriptomics. Plant Reprod. 2015; 28(2): 73–89. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSlotkin RK, Vaughn M, Borges F, et al.: Epigenetic reprogramming and small RNA silencing of transposable elements in pollen. Cell. 2009; 136(3): 461–472. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang XJ, Gaasterland T, Chua NH: Genome-wide prediction and identification of cis-natural antisense transcripts in Arabidopsis thaliana. Genome Biol. 2005; 6(4): R30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhan S, Lukens L: Protein-coding cis-natural antisense transcripts have high and broad expression in Arabidopsis. Plant Physiol. 2013; 161(4): 2171–2180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang X, Lii Y, Wu Z, et al.: Mechanisms of small RNA generation from cis-NATs in response to environmental and developmental cues. Mol Plant. 2013; 6(3): 704–715. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30668", "date": "21 Mar 2018", "name": "Hailing Jin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript by Qin et al. addresses an important question in the gene regulation field: How cis-natural antisense transcripts (cis-NATs) are co-regulated within the same cell. Although many genome-wide studies on cis-NATs have been reported in different species, most expression analysis was based on data from bulk samples that include many different cell types. The conclusions from those studies may not be entirely accurate because it is not clear if the two transcripts of each cis-NAT pair indeed express within the same cell. The authors of this study used a unique system – pollen for the analysis because pollen only has two cell types, sperm and vegetative cells, which allows easy identification of NATs that expressed in the same cell type. They identified 872 potential functional cis-NAT pairs in pollen. Microarray expression analysis identified 72 and 56 pairs were potentially functional in sperm and vegetative cells, respectively, as both adjacent genes were expressed. In addition, 271 pairs and 145 pairs for which only one was expressed in sperm or vegetative cell, respectively. It is possible that one of the transcripts within a pair that is expressed may suppress the expression of the other transcript in the same cell. Furthermore, 739 pairs of cis-NATs generated sRNAs and 35 cis-NATs pairs had sRNAs from the overlapping region, suggesting that sRNAs play a role in the co-regulation of NAT genes.\nCo-regulation analysis of cis-NATs within the same cell is an important but often overlooked criteria, this study provided an excellent example to take the localization of gene expression into consideration, which not only identified a set of pollen-specific functional cis-NATs and sRNAs for future study, but also help us understand the regulatory mechanism of cis-NATs. Thus, I support this manuscript to be indexed without delay.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "33361", "date": "10 May 2018", "name": "Robert A. Martienssen", "expertise": [ "Reviewer Expertise Epigenetics and small RNA in plants" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe have authors addressed an interesting question regarding gene regulation in vegetative and sperm cells in pollen by cis-natural antisense transcripts (cis-NATs). Qin et al. explain how cis-NATs are generated and their importance for plant development, pathogen resistance, stress response, homeostasis and fertilization. The main question for this work regards the discrepancy that may be occurring in previously published data, as most analysis were performed on bulked samples containing different types of cells leading to possible misassignment of cis-NAT pairing. cis-NATs should be co-expressed in the same cell, as the authors described, so the analysis should be done at the least on the same cell type. To address this issue, the authors used RNA-seq and microarray publicly available data from vegetative and sperm cells from Arabidopsis thaliana pollen in order to investigate cell-specific cis-NATs pairs, using specific parameters avoiding possible mispairing. The authors identified possibly functional cis-NATs in pollen, 56 pairs in the vegetative cell and 72 in sperm cells, also, the authors screened for small RNAs generated from the pairing regions, finding that 35 out of 739 pollen cis-NATs from the overlapping region, suggesting a regulatory function. It would be interesting to summarize in the text if any of these were in sperm vs VN.\n\nFunction analysis is yet to be addressed, as the authors pointed out, to confirm the biological role of cis-NATs in plant development, stress response, pathogen resistance, homeostasis and fertilization. But the authors choice to survey pollen grains where cell specificity is known could be key to understanding molecular mechanisms regarding fertilization and heredity.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "33655", "date": "16 May 2018", "name": "Jules Deforges", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this study, the authors have addressed an important question often neglected regarding the regulation of gene expression by cis-Natural Antisense Transcripts. Cis-NATs usually identified by sequencing of tissues containing many different cell types are assumed to potentially regulate their cognate coding genes co-expressed in the same tissue. However, direct evidence that both transcripts of the pairs are actually expressed in the same cells is often missing.\nBy taking advantage of RNAseq, micro-array as well as sRNA data from pollen, a tissue containing only 2 cell types, the authors identified 72 cis-NAT pairs for which both transcripts were expressed in Sperm cells and 56 in vegetative cells. In addition, they identified sRNA falling in the overlapping region of 35 cis-NAT pairs out of 739 in pollen, providing insight about the functional role of these cis-NATs.\nQin et al. provide useful data about cis-NAT expression in sperm and vegetative cells in pollen. They also provide insight about their potential function in sRNA production, contributing to a better understanding of the mechanisms of gene regulation by cis-NATs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-93
https://f1000research.com/articles/6-1698/v1	18 Sep 17	{ "type": "Research Article", "title": "Maize phyllosphere microbial community niche development across stages of host leaf growth", "authors": [ "Heather C. Manching", "Kara Carlson", "Sean Kosowsky", "C. Tyler Smitherman", "Ann E. Stapleton", "Heather C. Manching", "Kara Carlson", "Sean Kosowsky", "C. Tyler Smitherman" ], "abstract": "Background: The phyllosphere hosts a variety of microorganisms, including bacteria, which can play a positive role in the success of the host plant. Bacterial communities in the phylloplane are influenced by both biotic and abiotic factors, including host plant surface topography and chemistry, which change in concert with microbial communities as the plant leaves develop and age. Methods: We examined how the Zea mays L. leaf microbial community structure changed with plant age. Ribosomal spacer length and scanning electron microscopic imaging strategies were used to assess microbial community composition across maize plant ages, using a novel staggered experimental design. Results: Significant changes in community composition were observed for both molecular and imaging analyses, and the two analysis methods provided complementary information about bacterial community structure within each leaf developmental stage. Conclusions: Both taxonomic and cell-size trait patterns provided evidence for niche-based contributions to microbial community development on leaves.", "keywords": [ "epiphytes", "plant host", "community assembly", "Zea mays L.", "ecological niche", "scanning electron microscopy" ], "content": "Introduction\n\nThe phyllosphere, the microbial community inhabiting the surface of plant leaves1, can play important positive roles for the plant partner. For example, disease resistance is increased when supportive bacteria are present2,3 and increased host plant growth in the presence of specific community members has been documented4–6, along with a positive effect of overall increased microbial diversity7. Leaf habitats are also extremely important for the microbial community, as this habitat is quite large in surface area globally. In temperate regions seasonal growth and annual agricultural plantings lead to repeated opportunities for colonization by adapted microbes8,9.\n\nDeterminants of community assembly/succession in microbial systems include biotic and abiotic factors, often classified into different community assembly rules10. Typically random or neutral stochastic models are tested against abiotic effect models, which are called environmental, or, more recently, habitat filtering rules. Environmental filtering can produce different patterns depending on scale11. As recommended by Kraft et al. (2015) we will refer to environmental filtering as habitat filtering when there is no separate test for abiotic and biotic effects. A second class of assembly rules addresses the interactions of organisms, by either facilitation or competitive exclusion. These niche interactions are often modeled separately from abiotic effects, or abiotic effects are assumed to happen first.\n\nCommunity assembly and succession processes are typically inferred from diversity patterns in community samples12. The number of different taxa (alpha-diversity), the difference in the composition of taxa between replicates (beta-diversity) and the proportion of co-occurring taxa all provide evidence for these different community-generating mechanisms. Niche construction is inferred from a pattern of decreasing beta-diversity, increasing co-occurrence, and increasing richness, while habitat filtering is characterized by decreasing beta-diversity with no trends in co-occurrence or richness13,14. In contrast, neutral community assembly would exhibit increased richness (provided immigration rates are maintained), with unchanged beta-diversity and co-occurrence levels. Once we have measurements of the various components of diversity, we can compare observed trends to these expectations. The combination of increasing/decreasing trends would then support or fail to support a particular mechanism of community succession. Both trait-based (morphological, in our study) and taxonomic measures of diversity can be compared to the expected trends15,16. For microbial systems, cell size is one of the few traits that is measurable at the microscopic scale; other functional traits, such as metabolic activity, are more often examined at the molecular scale, by whole-community DNA sequencing, proteome analysis, or metabolite profiling.\n\nPrevious analyses of controlled inoculation phyllosphere community development indicate that habitat filtering is important, with niche effects less often detected across time, though all previous studies have used seasonal or through-time designs that confound exposure time and plant development. Pre-colonization reduces immigrant success8,17, suggesting biotic competition is key, though high beta-diversity in replicate samples from both pre-colonized and newly inoculated communities suggested that there are many options for species that arrive first to succeed. Several crop species have been tested for seasonal patterns of phyllosphere community development. Microbial populations increased more rapidly on younger leaves of lettuce18, and alpha-diversity increased over the first few weeks then plateaued19, which provides evidence that habitat filtering was a contributor to community succession later in the season. In apple flowers, species richness increased early in the season and beta-diversity dropped, then rose20. About half the apple flower microbial species had significant co-occurrence relationships and the numbers of co-occurrences increased and decreased over time. This pattern supports niche construction as a key mechanism for apple flower colonization. As in apple flowers, across-season dicot crop phyllosphere samples became less diverse (alpha-diversity went up then declined). In the crop samples, more shared and plant-specific taxa were found later in the season21, again supporting development of specific community structure. In contrast, Arabidopsis epiphyte beta-diversity increased with time22, providing stronger support for stochastic effects in this system. The history of annual opportunities for new colonization is reflected in the processes for dispersal and succession on plant leaves. Colonization of new growth in the presence of older leaves may proceed differently than colonization from distant source materials. The contributions of abiotic changes and host leaf age are confounded when sampling occurs through time, as in seasonal sampling, thus necessitating alternative experimental designs and/or additional phylogenetic or functional trait information to examine the roles of these assembly processes. Sampling serially across time, as all prior studies have done, could confound short and long-term trends and does not enable differences in plant host to be distinguished from inoculation processes19,20. Therefore, for this study host plant seeds were planted at different times and sampled once.\n\nHow can leaf microbial communities be measured? Molecular methods for detection of species using ribosomal fragments or sequences have become increasingly common20,21,23. Microscopy can be used to detect bacteria (and fungi) on corn leaf surfaces24, and surface bacteria can be followed with reporter genes25 as well as by direct enumeration. Microscopic or particle-based enumeration methods are especially useful for microbial communities where we have limited information on traits and horizontal gene transfer is likely26. For this study we used both a molecular approach to measure relatedness-based (taxonomic) microbial community composition, and direct enumeration of scanning electron micrograph (SEM) images to measure cell-size trait changes.\n\nOur specific questions concerned the pattern of community development – how community succession varies with plant age, and whether the microbial community was primarily shaped by microbe-host interactions (environmental or more generally, habitat filtering) or by microbemicrobe interactions (niche construction). We found complex patterns in the maize leaf microbial community structure even across the short series we sampled, with niche construction and stage-specific increasing-decreasing diversity trends observed.\n\n\nMethods\n\nZea maysL. B73 inbred seeds obtained from the Maize Genetics Cooperation Stock Center (http://maizecoop. cropsci.uiuc.edu/) were planted using a hand held planter between April 26th and June 15th 2009 at the Central Crops Research Station in Clayton, NC (http://maizecoop.cropsci.uiuc.edu/). All necessary permits were obtained for the described study, which complied with all relevant regulations; permits were managed by the research station staff. Approximately twenty seeds were planted each date, with plantings between four and eleven days apart in a single field. All plants within a developmental stage were planted in the same row, bordered on either side by older and younger stages, then by additional crops, grass borders, and tree-shrub hedgerows.\n\nAll ten replicate leaf samples were collected at once on July 15, 2009. Both scanning electron microscope (SEM) and ribotype (ARISA) samples were obtained from tips of separate central leaves from ten plants at each developmental stage. SEM samples were approximately five centimeters square and were cut along the leaf mid-rib near the tip (Figure 1). Sections were immediately fixed, by immersion in a solution of 2.5 percent glutaraldehyde in 0.1 M Sorenson’s phosphate buffer at a pH of 7.1.\n\nEach plant in the drawing represents 10 plants from one of seven developmental stages; the experimental design had n=10. Small squares represent SEM sampling areas and large squares represent ARISA sampling areas. All samples were taken from center leaves within each developmental group, and both samples per plant were taken from leaves of comparable position based on height.\n\nLeaf wash samples for ribosomal DNA analysis were then collected from the same ten plants, using adjacent leaf numbers and a similar portion of the leaf tip (Figure 1). Approximately 10 cm of leaf was cut, submerged in buffer (0.2mM Tris pH 7.5, 0.02 mM EDTA, 0.00012X Triton X100), agitated vigorously and removed using the method described in 27. Leaf washes were stored on dry ice while in the field then placed in a -70 degrees C freezer for long-term storage. One mock control (where no leaves were placed in the tubes) and one wash control (SEM sample from washed leaf to check for washing effectiveness) was taken for each stage.\n\nExcess glutaraldehyde was removed from leaf SEM samples by rinsing for 30 minutes in 0.1 M Sorenson’s phosphate buffer followed by an additional 30 minutes in distilled water. Sections were then dehydrated in an acetone series (50, 70, 90, 100 percent), each lasting 30 minutes, followed by a 30-minute treatment in hexamethyldisilazane. Sections were then allowed to air dry before being attached by the lower leaf surface to stubs using double-sided sticky tape. One leaf section chosen arbitrarily from each plant was sputter coated with 10 nm of platinum/palladium and observed in the SEM at 5 kV with a spot size of 3. The SEM procedure was tested prior to sampling using young (approximately 25 cm tall), greenhouse grown maize plants and Pseudomonas aureginosa28.\n\nUsing the SEM stage control, five evenly dispersed random images (quadrants) per plant were taken at 1000x and 2000x for 35 sections (35 plants) each to give two viewpoints by which to count bacteria. All images are available from the image repository BisQue via the iPlant/CyVerse Data Commons, at doi: 10.7946/P2WC77.\n\nFor the DNA analysis, leaf washes were syringe filtered using previously developed methods29 (videos of the method are available upon request) and microbial DNA was extracted using an UltraClean Microbial kit according to the manufacturer’s instructions (MoBio Laboratories, Carlsbad, CA). An equal volume of each DNA sample was amplified to ensure fair representation of the epiphytes collected.\n\nTwo separate, nested PCR reactions were performed on experimental samples as described by Cardinale et al.30, along with four positive controls from a mixture of DNAs from cultured bacteria (Carlson, 2015) and four negative (nuclease-free water) controls. For the initial PCR step, a master mix was made to include: Q5 master mix (New England Biolabs, Ipswich, MA), ITSF primer (5’-GTCGTAACAAGGTAGCCGTA-3’), LD primer (5’-CCGGGTTTCCCCATTCGG-3’), and nuclease-free ultrapure water. Ten microliters of master mix was added to sterile PCR tubes followed by experimental and sample controls. The samples and controls were then run on an iCycler (BioRad, Hercules, CA) PCR machine with the following settings: 1X 95C for 30 seconds, 35X (95C for 30 seconds; 61.5C for 30 seconds; 72C for 1 minute, 30 seconds), 1X 72C for 10 minutes. For the second PCR step, a second master mix was made with the following: Q5 master mix, ITSreub primer (5’-GCCAAGGCATCCACC-3’), which contained a fluorescent HEX marker for fragment analysis in a Genetic Analyzer, SD primer (5’-TGCGGCTGGATCCCCTCCTT-3’), and nuclease-free water.\n\nSamples were run on the Applied Biosystems Genetic Analyzer (ABI, Inc, Carlsbad, CA) and analysis of PCR product length was carried out using the instrument software. For each sample, two microliters of PCR product and 13 microliters of GS500-Rox plus HiDi (ABI, Inc, Carlsbad, CA) were combined. The samples were denatured at 94C for ten minutes, placed on ice for two minutes, then placed into the analyzer and the program started.\n\nFor each SEM quadrant image, Adobe Photoshop was used to manually outline each bacterial organism present (examples are shown in Figure 2). All single bacteria present in the image were outlined; bacteria were identified by size and smoothness of circumference, as originally described by Davis24. Bacterial areas were measured using Image Pro Plus (Media Cybernetics, Inc., Rockville, MD, USA) and recorded for each quadrant picture that was taken. Image Pro Plus sized bacteria that were touching other bacteria as one large organism, therefore as many bacteria as possible were outlined in the clusters without outlining touching bacteria. Each image was individually measured using calibration settings based on the magnification used to take the image. Each image analyst completed analysis of a training image set and the coefficient of variation of their analyses was checked to ensure that it was less than 5 percent.\n\nBacterial sizes acquired from all images were binned using methods described by Shimazaki et al.31. Bin sizes were assigned to the microbial samples as size-class units (SCU) and a microbial community profile was created for the samples using the RiboSort package in R32.\n\nA) Typical image after 30 days of growth in the field. B) 72-day image, with leaf hair and stomata clearly visible. C) Typical image after 80 days of growth in the field.\n\nFiles from the Genetic Analyzer were opened in Peak Scanner 2 software (http://www.appliedbiosystems.com) using Analysis Method=Sizing Standard-PP, and Size standard=GS500. Only the green (hex-primer-labeled) peaks were used to determine level of bacterial abundance. The size and heights of the green peaks were calculated using the ROX GS500 standard. The individual output files were assembled into a single microbial profile file using the RiboSort package32 using the script, RiboSort(data,dataformat=\"standard\",dye=\"G\", output=\"proportions\",zerorows=FALSE,repeats=1, mergerepeats=\"presentinall\"). This program automatically bins the peaks to generate abundances; we used the default threshold of 50 fluorescence units as recommended by the program authors.\n\nThe OTU (operational taxonomic unit) abundance matrices output from Ribosort were analyzed by Permdisp and Permanova33–35 using Primer-E software v636. Overall square root transformation and Bray Curtis similarity resemblance were applied to the initial abundance data within Primer-E before Permanova and Permdisp tests were run. The P-values from the program were Bonferroni-corrected. Primer-E SIMPER analysis was used to compute the number of OTU contributing to differences between stages. Primer-E functions were used to compute diversity measures and taxa numbers for each sample (Supplementary Dataset).\n\nThe evenness (Pielou’s J’) of OTU and SCU for each sample was computed using Primer-E v636. The values of J’ for OTU and SCU were compared across plant stages by rank-transformed analysis of means using the routines in JMP Pro v11 (SAS Inc, Cary, NC) with P-values adjusted for multiple comparisons. For the rank-transformed analysis of means the P-value threshold was set at the Bonferroni-corrected value of P less than 0.007.\n\nThis rank-transformed analysis of means is not sensitive to heteroschedasticity and is more powerful than other nonparametric tests when the data distribution is heavy-tailed37. Nonparametric pairwise comparisons of evenness between stages were done using the Steel-Dwass multiple-comparison method. The distributions of SCU areas in each stage of plant growth were also computed with JMP Pro v11.\n\nPairwise analysis was completed using the R package Co-Occur38; this package allows determination of the number of OTU/SCU that occur together in the same sample day at a probability above random, along with the number of taxa that are not found together (negatively correlated taxa) above the random-pair threshold. The analysis code, input and output files are available at https://github.com/skosowsky/Cooccur. We created network visualizations of the co-occurring OTU and SCU with Cytoscape (v. 3.4) and compared networks using the Cytoscape plug-in app DyNet (v1.0)39. Intensity of node connection change across the seven stages is reflected in the intensity of the color of the DyNet-analyzed nodes.\n\nThe number of OTU and SCU present within each sample was computed using Primer-E v6 (abbreviated as S, also known as Chao1). The number of OTU and SCU compared to the overall mean across plant stages was analyzed using rank-transformed analysis of means via routines in JMP Pro v11 (SAS Inc, Cary, NC), with P-values adjusted for multiple comparisons. For the ranktransformed analysis of means the P-value threshold was set at the Bonferroni-corrected value of P=0.007. This rank-transformed analysis of means is not sensitive to heteroschedasticity and is more powerful than other nonparametric tests when the data distribution is heavytailed. Nonparametric pairwise comparisons between stages were carried out with the Steel-Dwass multiple-comparison method within JMP.\n\n\nResults\n\nOur experimental design allowed for a single microbial sampling that was not confounded by differential exposure to recent weather events, with sampling for molecular and microscopic analyses in each plot (Figure 1). Molecular analysis of community ribotype composition (ARISA) and image analysis of microbial community size cell assemblage size classes (by scanning electron microscopy, SEM) provided two complementary views of community assembly processes.\n\nARISA community composition and beta-diversity. Microbial community composition changed over time in bacterial ribosomal taxa (Permanova P-value of 0.026), as measured by differences in operational taxonomic units (OTU). The Permanova pairwise results for bacterial ribosomal OTU summarized in Table1 and with additional details in Supplementary Table 1 and Supplementary Table 2 indicate that, specifically, day 30 and day 80 were significantly different from each other in community composition. Thus we conclude there was a difference in either the richness or arrangement of the communities’ ribotypes in the earliest and latest stages of community development. To determine which aspect of community structure contributed to the difference detected by Permanova, we examined the dispersion as a measure of beta-diversity, i.e. the amount of range in the set of species present across replicates within each time. There was no significant change in the dispersion in the ribosomal OTU datatype across stages (Permdisp P=0.445, details in Supplementary Results). To further analyze the ARISA community structure, we compared the evenness of species distributions across stages.\n\nSignificance groups with different letters are significantly different by pairwise test with a Bonferroni multiple-test-correction threshold of P=0.001315.\n\nEvenness is a measure of the range of OTU abundances in a sample unit; we used the Pielou’s J’ measure of sample evenness for comparison of stage evenness values. Evenness values were not normally distributed and were heavy-tailed (with kurtosis less than 3), so nonparametric tests were used for analysis of evenness differences across stages. Evenness was significantly different (P=0.0354) between stages 30 and 80 in the ARISA datatype in nonparametric pairwise tests (see Supplementary Results section 1.b for details), with the evenness value lower in the latest stage (i.e. a few OTU are more abundant in the stage 80 samples, compared to a more similar set of abundances in the first samples), though overall the evenness averages are not significantly different from the overall mean across all the sample days (Supplementary Results section b). This pairwise-significant evenness difference in important OTU abundance ranges in the earliest and latest sample dates is also visible in a SIMPER analysis of ARISA OTU, with the top contributions to the day 30 OTU more evenly spread (for day 30: OTUs with 31, 19, 11, 11, 8, 6, 4 percent) as compared to day 80 (with OTUs of 50, 19, 10, 9, 5 percent, Supplementary Table 4).\n\nSEM community composition and beta-diversity. In contrast to the ARISA datatype, in the SEM-based size-class unit (SCU) datatype the beta-diversity significantly decreased in the day 41 samples relative to the day 48 samples (Permdisp P=0.004, Table 2, details in Supplementary Table 3). This beta-diversity difference confounds Permanova tests, so we cannot further compare SEM community compositions across stages with that method. The significant SEM beta-diversity community change tells us that there is an important successional process at this specific stage relative to the earlier and later samples. Comparison of the Pielou’s J’ evenness values in the SCU datatype using nonparametric tests indicated that day 72 had significantly lower evenness of size-classes relative to the overall mean (Supplementary Results section 2), with pairwise tests showing that day 72 was significantly lower in evenness than day 80 (Supplementary Results 2.b). Pairwise comparisons among all other days were not significantly different.\n\nSignificance groups with different letters are significantly different by pairwise test with a Bonferroni multiple-test-correction threshold of P=0.001315.\n\nThe pale blue area is the confidence interval (UDL and LDL) above and below the overall mean. Rank-means for each stage are points at the tip of the drop-down bars. Significant means (adjusted P<0.05) have red line tips, while non-significant means within the confidence interval have green tips. A) ARISA mean differences with transformed ranks. B) SEM mean difference of transformed ranks of taxa.\n\nWe compared the number of OTU present in each sample to the overall mean using nonparametric comparisons suitable for non-normal, heavy-tailed distributions (Figure 3). The number of OTU – the richness – increases significantly in the ARISA datatype at day 62 (this mean is above the confidence interval and is thus indicated with a red dot in Figure 3 a), with earlier and later samples showing no significant difference from the overall mean richness (means indicated with green dots) and no pairwise differences in means compared to each other with nonparametric tests (Supplementary Results section 1a). In contrast to the ARISA datatype pattern, the number of different SCU present in the size-class datatype is significantly lower at day 48 (Figure 3b), while all other stages are within the confidence limit for the overall mean richness. Nonparametric pairwise comparisons of SCU indicated that day 48 was significantly lower than all later-stage samples and that days 72 and 80 had significantly higher SCU means present than day 30 (Supplementary Results section 2a).\n\nFor the ARISA datatype, there were three OTU found in all stages of community development; these were OTU9, OTU22, and OTU24, which comprised 1.5 percent of the total number of OTU. For the SEM datatype, there were 13 SCU (5.7 percent of the total); a visual display of the proportions of all 13 across the seven sample dates is shown in Supplementary Figure 3.\n\nTo further examine community assembly processes we looked at trends in the co-occurrence of pairs of species in both phyllosphere datatypes. We examined these patterns in our data to identify ways that community composition could differ. Positive co-occurrence is defined as species occurring together more often than random (and thus having potential mutualistic interactions), while negative co-occurrence values indicate that the species pair is found together less often than the random expectation (potential antagonistic interactions). Co-occurrence patterns changed across plant stages in both ARISA (Figure 4) and SEM (Figure 5). Participation of certain OTU and SCU in varying interactions across multiple stages is indicated by the red color intensity and number of overall edge interactions in the DyNet networks (Figure 4b and Figure 5b). In the ARISA datatype, highly variable co-occurring OTU (intense red color in Figure 4b) had both positive and negative interactions with other community members. In contrast, the SEM datatype exhibited more positive interactions connecting highly dynamic nodes (intense red nodes in Figure 5b). There were more positive than negative significant co-occurrences in both the ARISA and SEM datatypes (Figure 4 and Figure 5, edge arrows with points). The number of co-occurrences increased with increasing plant growth time in each datatype, though not at the same stage; as seen in Figure 4, ARISA had the largest number of network nodes at day62 and for SEM (Figure 5) the largest number of nodes was day72. The overall proportion of co-occurring pairs was similar in each datatype (Table 3), with a minority of microbial types found to have significant co-occurrence partners. Specific OTU and SCU identifiers are individually displayed in stacked plots that show details about which OTU occurred in more than one pair within Supplementary Figure 1 and Supplementary Figure 2.\n\nIf there were no significant co-occurring OTU, then that sample stage was not shown. Negative interactions between OTU nodes are shown as edges with a barred arrow tip and positive correlations between OTU are indicated with pointed arrows. a) Significant co-occurring OTU from each sampled stage. b) Results of DyNet analysis of co-occurring OTU most changed across stages. Only significant co-occurring OTU (as shown in part a) were analyzed using DyNet. Intensity of the red color of nodes indicates amount of change across stages, with darker color indicating the most ‘rewiring’ across stages.\n\nIf there were no significant co-occurring SCU, then that day was not shown. Negative interactions between SCU nodes are shown as edges with a barred arrow tip and positive correlations between SCU are indicated with pointed arrows. a) Significant co-occurring SCU from each sampled stage. b) Results of DyNet analysis of co-occurring SCU most changed across stages. Only significant co-occurring SCU (as shown in part a) were analyzed using DyNet. Intensity of the red color of nodes indicates amount of change across stages, with darker color indicating the most ‘rewiring’.\n\n\nDiscussion\n\nOur experimental design was selected to avoid confounding by differential exposure to recent weather events. This design, which has recently been recommended by Shade et al.(2013) and by Williams et al.(2013), allowed us to examine the contributions to community assembly due to dispersal, filtering and niche-related processes. Comparisons of presence-absence patterns to presence-only richness within a datatype are complementary analyses, as the presence-absence matrix allows for overall tests of community difference (Permanova-based, 33) and tests for beta-diversity difference as difference in homogeneity of variance (Permdisp, 34). Presence-only lists of taxa or size-classes allow examination of the richness dimension of the microbial communities, abstracted away from other structural differences.\n\nAre two datatypes better than one? We measured a morphological trait (microbe size) and a relatedness (ribosomal length difference) metric, and the two datatypes were not especially similar at specific time points, making them complementary rather than redundant; we infer that predictive power from one datatype to the other would be low. The differences between datatypes suggest that size-classes may include different taxa, in other words our size-classes may not have a phylogenetic signal. Some recent work in phytoplankton and soil microbial communities contrasts with our results, showing that traits can be conserved across taxonomic groupings26,40; generally conservation of traits and evolutionary relatedness is likely to depend on the genetic architecture of the phenotypic trait41. The relationship between metabolic and functional traits and phylogenetic signal is as yet unclear for microbial communities that have substantial levels of gene transfer42,43. Complementary information provided by two (or more) datatypes is thus recommended for future phyllosphere experiments.\n\nMisclassification of ribosomal spacer lengths to different taxa when they are in fact due to population differences, and misclassification of large bacterial cells in a size-class separate from their smaller offspring, would have consequences for trends across samples if the misclassification probability were different at the different stages of community development. In our study, the underlying assumption is that our probability of misclassification is the same across stages. For size-classes, misclassification could increase with time due to growth of cells, resulting in a continuous increase in the number of SCU over time. We do not see this trend in our data, so from our study there is no evidence of confounding misclassification. We cannot rule out misclassification that occurs only at specific stages using our experimental design. We suggest that sample processing for future experiments include inline size separation so that size-classes, ribosomal data, and molecular/metabolic data are collected as true multivariate data in each sample. Image information such as our scanning electron micrographs or fluorescence-based microbial images could also be analyzed for patterns that could illuminate resource use traits and specific cell-shape-based mutualisms44.\n\nTrends in richness, co-occurrence trends, and beta-diversity changes together define overall community assembly mechanistic contributions, with habitat filtering characterized by unchanged richness, unchanged co-occurrence frequency, and decreasing beta-diversity. Niche development would be observed as a combination of increasing richness, increasing co-occurrence, and lowered beta-diversity. Finally, a neutral pattern – with no change in immigration probability over time – would exhibit trends toward increasing richness, unchanged co-occurrence, and unchanged beta-diversity. Comparison of recent phyllosphere analyses and our work using this framework emphasizes the role of niche development as a mechanism of community assembly.\n\nFirst we compare our results with recent work in apple flower microbial communities, where beta-diversity was initially at relatively high levels, decreased, then increased over the remainder of the season; richness (measured as phylogenetic diversity in the apple flower work) increased then leveled off, and finally co-occurrences showed early increase then decline and rebound toward higher levels over the season20. To summarize, early in the apple flowering season the diversity patterns support niche development mechanisms, and later in the season both habitat filtering and niche development patterns were seen. In our study of maize leaf microbial epiphytes we also found that there was no overall temporal trend. Our leaf ARISA pattern of diversity change was not especially similar to the apple flower changes; for example, we found no significant change in beta-diversity, while beta-diversity declined then rose over the later part of the season in apple flowers. Our ARISA richness and the apple flower phylogenetic diversity also differed, with apple flower phylogenetic diversity increasing then leveling off, while on maize leaves richness increased only in one later growth stage. The evenness in both our ARISA and apple flower phylogenetic diversity varied; in apple flower the ‘mid’ stage had a lower evenness while in our ARISA dataset the day 80 evenness was lower than in day 30. Co-occurrences in our study increased in mid-season, as did the apple flower communities in the early flower stage. Overall, the early season patterns in the across-season apple flower microbial community analysis were the most similar in community assembly to our maize leaf staggered-stage analysis. In addition to the apple flower study, in a recent study of three different crop leaf phyllosphere seasonal patterns, Copeland et al.21 found that alpha-diversity increased then decreased across the season, with more shared species later in the season. In all three crops, beta-diversity (as measured as Unifrac distance for leaf-specific microbes) decreased across season, though not evenly; the beta-diversity decrease was more prominent later in season and at one time-point was higher during the later phase. Co-occurrences were not analyzed in this study, so these crop study results are consistent with niche and/or habitat mechanisms.\n\nCore microbial communities in crops are those that are either recruited early and retained, or continually input across the season. These are often found by presence45, as in our analysis of ARISA and SEM core microbial community members. We found a relatively small proportion of core OTU and SCU; other phyllosphere studies such as on apple flowers20 found more (for apple flowers the generalist category was 14 percent, for example). Our core set should be considered as a first description, as we did not consider absences or compare our core set to air or soil samples. Instead, our core set focused on microbial community members that colonized leaves of all developmental stages.\n\n\nConclusions\n\nOur ARISA taxonomic diversity patterns and microbial cell size trait datatypes exhibited different specific patterns of diversity, but there was similarity in trend overall, as both datatypes support niche process contributions to community assembly. Niche (deterministic) processes have recently been found to be important in soil microbial communities46,47. In lakes, microbial generalists found at multiple spatially separated sites had a better fit to neutral stochastic models, with specialists exhibiting a more deterministic pattern48. As leaf surface communities are quite different than air or soil communities9, and the number of core species in our inventory is low, we suggest that many of the maize leaf microbial community member that we detect are specialists. Our staggered experimental design highlighted the role of microbe-microbe interactions in leaf microbial community development. We thus suggest that future experiments use staggered experimental designs and multivariate metabolic, genic, and size-trait data collection.\n\n\nData availability\n\nDataset 1: ARISA and SEM taxonomic abundance data. DOI, 10.5256/f1000research.12490.d17751649\n\nDataset 2: Detailed description of column headers, row labels and cell content for Dataset 1 and Supplementary materials. DOI, 10.5256/f1000research.12490.d17751750\n\nFiles in the CyVerse/iPlant Data Commons repository are available from http://datacommons.cyverse.org/browse/iplant/home/shared/commons_repo/curated/Stapleton_MaizeLeafMicrobesByLeafAge_2015, with DOI, 10.7946/P2WC77. This repository includes the following individual files:\n\nSupplementarySEMfiles Folder\n\nbinnedSEMSizes.csv\n\nreadmeSEMfolderfilemetadata.txt\n\nSEMImages Folder and Subfolders\n\nSEMoverlays Folder\n\nSEMribosortinputfiles Folder\n\nSEMRiboSortoutputfiles Folder\n\nSupplementaryARISAfiles Folder\n\nARISAfilenamemetadata.csv\n\nARISARibosortinput Folder\n\nARISARibosortoutput Folder\n\nARISAruns Folder and SubFolders\n\nreadmeARISAfolderandfilemetadata.txt\n\nSource data files formatted for CytoScape visualization are available at https://github.com/AEStapleton/plantstage.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to A. Ulloa-Bacillio, UNCW and J. Geyer, UNCW for assistance with SEM image analysis, to the UNCW microscopy facility for training and equipment support, and to the staff of the Central Crops research station for experimental field management.\n\n\nSupplementary material\n\nSupplementary Figure S1: a-n Arisa Plots of Signifcant Co-occurring OTU Pairs.\n\nClick here to access the data.\n\nSupplementary Figure S2: a-n Plots of SEM datatype co-occurrence at each growth stage.\n\nClick here to access the data.\n\nSupplementary Figure S3: SEM Core SCU.\n\nClick here to access the data.\n\nSupplementary Results.\n\nClick here to access the data.\n\nSupplementary Table 1.\n\nClick here to access the data.\n\nSupplementary Table 2.\n\nClick here to access the data.\n\nSupplementary Table 3.\n\nClick here to access the data.\n\nSupplementary Table 4.\n\nClick here to access the data.\n\n\nReferences\n\nVacher C, Hampe A, Porté AJ, et al.: The Phyllosphere: Microbial Jungle at the Plant-Climate Interface. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavis DG, Meola SM, Mullins JS: Extraneous Material on Plant Surfaces: An SEM Study. Weed Science. 1976; 24(3): 341–347. Reference Source\n\nRemus-Emsermann MN, Tecon R, Kowalchuk GA, et al.: Variation in local carrying capacity and the individual fate of bacterial colonizers in the phyllosphere. ISME J. 2012; 6(4): 756–765. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPortillo MC, Leff JW, Lauber CL, et al.: Cell size distributions of soil bacterial and archaeal taxa. Appl Environ Microbiol. 2013; 79(24): 7610–7617. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKadivar H, Stapleton AE: Ultraviolet radiation alters maize phyllosphere bacterial diversity. Microb Ecol. 2003; 45(4): 353–361. PubMed Abstract \| Publisher Full Text\n\nSmitherman CT: The Zea mays phyllosphere exhibits increases in abundance and decreases in diversity of bacteria through plant development. M S University of North Carolina Wilmington, 2010.\n\nStapleton AE, Simmons SJ: Plant control of phyllosphere diversity: genotype interactions with ultraviolet-B radiation. In Bailey MJ, Lilley AK, Timms-Wilson TM, and Spencer-Phillips PTN, editors, Microbial Ecology of Aerial Plant Surfaces. CAB International, Wallingford, 2006; 223–239. Publisher Full Text\n\nCardinale M, Brusetti L, Quatrini P, et al.: Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl Environ Microbiol. 2004; 70(10): 6147–6156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShimazaki H, Shinomoto S: A method for selecting the bin size of a time histogram. Neural Comput. 2007; 19(6): 1503–1527. PubMed Abstract \| Publisher Full Text\n\nScallan U, Liliensiek A, Clipson N, et al.: ribosort: a program for automated data preparation and exploratory analysis of microbial community fingerprints. Mol Ecol Resour. 2008; 8(1): 95–8. PubMed Abstract \| Publisher Full Text\n\nAnderson MJ: A new method for non-parametric multivariate analysis of variance. Austral Ecol. 2001; 26(1): 32–46. Publisher Full Text\n\nAnderson MJ: Distance-based tests for homogeneity of multivariate dispersions. Biometrics. 2006; 62(1): 245–253. PubMed Abstract \| Publisher Full Text\n\nAnderson MJ, Ellingsen KE, McArdle BH: Multivariate dispersion as a measure of beta diversity. Ecol Lett. 2006; 9(6): 683–693. PubMed Abstract \| Publisher Full Text\n\nClarke KR, Gorley RN: User manual/tutorial. PRIMER-E, Plymouth, 2006.\n\nNelson P, Wludyka P, Copeland K: The Analysis of Means: A Graphical Method for Comparing Means, Rates, and Proportions. SIAM Press, Philadelphia, PA, 2005. Publisher Full Text\n\nVeech JA: A probabilistic model for analysing species co-occurrence. Glob Ecol Biogeogr. 2013; 22(2): 252–260. Publisher Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–2504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBruggeman J: A Phylogenetic Approach to the Estimation of Phytoplankton Traits (1). J Phycol. 2011; 47(1): 52–65. PubMed Abstract \| Publisher Full Text\n\nMartiny JB, Jones SE, Lennon JT, et al.: Microbiomes in light of traits: A phylogenetic perspective. Science. 2015; 350(6261): aac9323. PubMed Abstract \| Publisher Full Text\n\nGoberna M, Verdú M: Predicting microbial traits with phylogenies. ISME J. 2016; 10(4): 959–967. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBoon E, Meehan CJ, Whidden C, et al.: Interactions in the microbiome: communities of organisms and communities of genes. FEMS Microbiol Rev. 2014; 38(1): 90–118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJi Z, Card KJ, Dazzo FB: CMEIAS JFrad: A Digital Computing Tool to Discriminate the Fractal Geometry of Landscape Architectures and Spatial Patterns of Individual Cells in Microbial Biofilms. Microb Ecol. 2015; 69(3): 710–720. PubMed Abstract \| Publisher Full Text\n\nShade A, Handelsman J: Beyond the Venn diagram: the hunt for a core microbiome. Environ Microbiol. 2012; 14(1): 4–12. PubMed Abstract \| Publisher Full Text\n\nCao P, Wang JT, Hu HW, et al.: Environmental Filtering Process Has More Important Roles than Dispersal Limitation in Shaping Large-Scale Prokaryotic Beta Diversity Patterns of Grassland Soils. Microb Ecol. 2016; 72(1): 221–230. PubMed Abstract \| Publisher Full Text\n\nPowell JR, Karunaratne S, Campbell CD, et al.: Deterministic processes vary during community assembly for ecologically dissimilar taxa. Nat Commun. 2015; 6: 8444. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiao J, Cao X, Zhao L, et al.: The importance of neutral and niche processes for bacterial community assembly differs between habitat generalists and specialists. FEMS Microbiol Ecol. 2016; 92(11): pii: fiw174. PubMed Abstract \| Publisher Full Text\n\nManching H, Carlson K, Kosowsky S, et al.: Dataset 1 in: Maize phyllosphere microbial community niche development across stages of host leaf growth. F1000Research. 2017. Data Source\n\nManching H, Carlson K, Kosowsky S, et al.: Dataset 2 in: Maize phyllosphere microbial community niche development across stages of host leaf growth. F1000Research. 2017. Data Source" }	[ { "id": "26099", "date": "18 Oct 2017", "name": "Kathryn M. Docherty", "expertise": [ "Reviewer Expertise Microbial ecology", "soil", "airborne", "biodegradation" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI reviewed the manuscript \"Maize phyllosphere microbial community niche development across stages of host leaf growth\" by Manching et al. published in F1000Research. I rated this article \"Approved with Reservations\".\nSummary: The researchers used a staggered experimental design to examine microbial community assembly processes on leaves of Zea mays. That is, they planted seeds at different times, and then collected samples on one date from 7 different growth stages of maize (Days 30, 41, 48, 58, 62, 72, 80). They examined leaf samples from 10 plants at each growth stage using two approaches. First, they examined microbial communities using SEM and categorized microorganisms into different size class units (SCUs). Second, they examined microbial communities using ARISA and categorized taxa into different operational taxonomic units (OTUs). Using microscopic and molecular approaches they found different results. Size-classes varied on days 41 and 48, first dropping in average size and then increasing in average size. On the rest of the dates, the size classes did not differ. OTUs were different between days 30 and 80, but for the rest of the time period, the communities did not differ significantly. To explore this further, the researchers used a network analysis approach to elucidate how either specific SCUs or OTUs were related to changes (positive or negative) in other SCUs or OTUs, respectively. They found that there were more positive co-occurrences than negative co-occurrences in both datasets, but that most individual data points did not have a significant co-occurrence pattern with other members of the dataset. They found a relatively small number of core SCUs and OTUs across the different time points of the dataset. They suggest that this may indicate that there are a number of specialists in the maize phyllosphere microbial community, and conclude that their results provide evidence for niche-based contributions to microbial community assembly on maize leaves.\nOverall, the manuscript provides novel information about changes in phyllosphere microbial communities during development stages of an important agricultural crop. The idea that plant leaves can be used for basic studies of microbial community assembly is interesting and the use of complementary datasets (size and composition) was a good approach, because it gets at 2 aspects of the community. There were a few issues with the manuscript that I believe need to be addressed before it is acceptable.\nWork is discussed appropriately in the context of the current literature\nNo, unfortunately the literature review needs to be updated. There are several important publications in this field that should be included, and this will require a re-write of the introduction and discussion sections. See: Redford et al.1 Kembel et al.2\nWhether suitable methods have been used\nThere are several things that are unclear about the choices for the experimental design. Please address:\n- Why were the specific day-intervals and developmental stage-dates chosen? Do they relate to specific physiological processes in maize development or were they chosen at random? Explain this choice further in the methods/results.\n- Planting all seeds for a particular treatment (i.e. development stage) in the same row sets the experiment up for statistical issues that are usually dealt with using a randomized block. In the current experimental design, one can interpret the results that (for example) day 30 and day 80 plant samples differ in OTU composition simply because the 30 day plants are subject to a different environmental variable than the 80 day plants (maybe soil nitrogen or light or water). Please re-do analyses using a randomized block and provide any information to suggest that there is no gradient that may account for significant differences across treatments.\n\n- The samples were collected and analyzed in 2009. At that time ARISA was a suitable method for this study, but since then, several other methods have been developed that could address the hypotheses more fully. For example, using predicted metagenomics pathways might indicate that there is a connection between community structure and cell size that was not demonstrated with ARISA. The authors need to address that this could be the case in the discussion and that further studies using these techniques could yield different results.\nI liked that the investigators included control DNA extractions. Were there any OTUs associated with the controls? How were those dealt with in ARISA processing?\nI thought that the network visualization approach was interesting, but it is really unclear what the authors are showing with Figure 5b. If it is just that the interconnections between SCUs gets really complicated, then I do not think a figure is necessary.\nSufficient information and source data have been provided to allow others to repeat every step of the work\nYes, I think there is sufficient information to repeat the work.\nThe conclusions are supported by the findings.\nNo, I think that there are a few things that need to be addressed in the discussion. First, please refer to the references I listed above and the articles that have cited them since, and re-think the discussion in that new framework. Second, the statistical issues of having all of 1 treatment in 1 row need to be addressed, otherwise all statistical results could be considered related to some unknown environmental gradient.\nSome minor comments:\nThe introduction is mostly about community assembly. However, to me, the really interesting thing about this study is that it is done using a major agricultural crop. Little is discussed about why it would be interesting to know about bacterial community assembly in this plant. Perhaps there is a context for plant productivity or agricultural disease that could be expanded to make the intro more specific to the study?\nI found the organization of the manuscript difficult to follow. Please reorder so that all SEM-related sections are together and all ARISA sections are together in the methods. Then follow whatever order you have chosen for the results.\nThe supplementary data tables are simply raw R output. Can you summarize the raw output in a supplementary table and then provide the raw output so that it is easier for the reader to interpret? It would be easier to interpret Tables 1 and 2 if the significances were just included in Figure 3 instead of in a separate table. (i.e. put the letters above each of the averages shown in Figure 3 to indicate significances)\nFigure 3 - the red/green dots are difficult to see (and I am not red/green color blind, so it would be even harder for 10% of our colleagues). Please adjust these so that they are more obviously different or have different shapes.\nSupplementary Figure 3 needs a legend that identifies what each of the SCU_IDs are and more information about the graph.\nOn page 9, the authors state that \"In the ARISA datatype, highly variable co-occurring…\". Please provide more of a definition of what you mean by \"highly variable\". Is this quantifiable?\n\nDiscussion Section - there are 3 different referencing styles used. Change for consistency.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3217", "date": "13 Dec 2017", "name": "Ann Stapleton", "role": "Author Response", "response": "Reply to Reviewer 1 Thank you for your insightful comments and suggestions! We have addressed each point in the revised manuscript, and we explain each below, with review comments in italic and author replies in regular font. I reviewed the manuscript \"Maize phyllosphere microbial community niche development across stages of host leaf growth\" by Manching et al. published in F1000Research. I rated this article \"Approved with Reservations\". Summary: The researchers used a staggered experimental design to examine microbial community assembly processes on leaves of Zea mays. That is, they planted seeds at different times, and then collected samples on one date from 7 different growth stages of maize (Days 30, 41, 48, 58, 62, 72, 80). They examined leaf samples from 10 plants at each growth stage using two approaches. First, they examined microbial communities using SEM and categorized microorganisms into different size class units (SCUs). Second, they examined microbial communities using ARISA and categorized taxa into different operational taxonomic units (OTUs). Using microscopic and molecular approaches they found different results. Size-classes varied on days 41 and 48, first dropping in average size and then increasing in average size. On the rest of the dates, the size classes did not differ. OTUs were different between days 30 and 80, but for the rest of the time period, the communities did not differ significantly. To explore this further, the researchers used a network analysis approach to elucidate how either specific SCUs or OTUs were related to changes (positive or negative) in other SCUs or OTUs, respectively. They found that there were more positive co-occurrences than negative co-occurrences in both datasets, but that most individual data points did not have a significant co-occurrence pattern with other members of the dataset. They found a relatively small number of core SCUs and OTUs across the different time points of the dataset. They suggest that this may indicate that there are a number of specialists in the maize phyllosphere microbial community, and conclude that their results provide evidence for niche-based contributions to microbial community assembly on maize leaves. Overall, the manuscript provides novel information about changes in phyllosphere microbial communities during development stages of an important agricultural crop. The idea that plant leaves can be used for basic studies of microbial community assembly is interesting and the use of complementary datasets (size and composition) was a good approach, because it gets at 2 aspects of the community. There were a few issues with the manuscript that I believe need to be addressed before it is acceptable. Work is discussed appropriately in the context of the current literature No, unfortunately the literature review needs to be updated. There are several important publications in this field that should be included, and this will require a re-write of the introduction and discussion sections. See: Redford et al.1 Kembel et al.2 These references discuss across-taxa patterns, which is not our focus (we use one genotype of the domesticated crop Zea mays), so we did not include them in our manuscript. We have added two additional sources (Redford 2009 and Dees 2015) to the introduction section to expand our coverage of seasonal pattern literature. Whether suitable methods have been used There are several things that are unclear about the choices for the experimental design. Please address: - Why were the specific day-intervals and developmental stage-dates chosen? Do they relate to specific physiological processes in maize development or were they chosen at random? Explain this choice further in the methods/results. These sampling intervals were chosen to capture maize leaf developmental stages (classically described as V(1 to n) for vegetative growth); each stage occurs at intervals of approximately one week and our sampling dates reflect this timing. We added a statement to the methods section explaining this. - Planting all seeds for a particular treatment (i.e. development stage) in the same row sets the experiment up for statistical issues that are usually dealt with using a randomized block. In the current experimental design, one can interpret the results that (for example) day 30 and day 80 plant samples differ in OTU composition simply because the 30 day plants are subject to a different environmental variable than the 80 day plants (maybe soil nitrogen or light or water). Please re-do analyses using a randomized block and provide any information to suggest that there is no gradient that may account for significant differences across treatments. The experimental design was chosen to fit agricultural practice, specifically maize field cultivation practice. This means that seeds are planted in rows with spacing dictated by planting equipment (30 in rows at this field station) and weed control (cultivation), fertilization, and pest control are carried out in those rows. Our experimental seeds were planted in adjacent rows, so nested spatial models that incorporate the physical distance between plants across the plots would be an ideal model to fit. However, spatial multivariate models with zero-inflated distributions for species counts are quite complex and we chose not to add this complexity to our model, as we have no evidence for spatial gradients within these short physical distances; for example, the soil type is the same (within the limits of our measurement instruments), as is the temperature and light regime. Our experimental design controls for field-level effects of environmental history such as recent rainfall, but cannot and does not control for developmental history of exposure to environmental factors in a field setting. Controlled environment chambers would be required to control for past environment effects; however, chamber experiments are much less agronomically relevant, so we chose to carry out field experiments. - The samples were collected and analyzed in 2009. At that time ARISA was a suitable method for this study, but since then, several other methods have been developed that could address the hypotheses more fully. For example, using predicted metagenomics pathways might indicate that there is a connection between community structure and cell size that was not demonstrated with ARISA. The authors need to address that this could be the case in the discussion and that further studies using these techniques could yield different results. We agree that functional gene and metabolic pathway information would be valuable, and we suggest some specific hypotheses to test using such information in the discussion section. An automated cell-sorting assay would be required to collect suitable multivariate paired metabolic/pathway and cell size data, and we encourage development of instruments of this type. I liked that the investigators included control DNA extractions. Were there any OTUs associated with the controls? How were those dealt with in ARISA processing? After optimization of the nested PCR protocol there were no OTU associated with the controls. We did find that the PCR master mix was important for consistent amplification and low false positive rates, so we included details of the PCR reactions within our methods section. I thought that the network visualization approach was interesting, but it is really unclear what the authors are showing with Figure 5b. If it is just that the interconnections between SCUs gets really complicated, then I do not think a figure is necessary. With assistance from the F1000 editorial staff we attempted to create interactive network graphics that allow readers to zoom and highlight, but it was not technically feasible at this time. Future analyses of succession might explore network modularity measures, as the networks we see in Fig. 5 appear to show modularity, especially in conjunction with future metabolic pathway analyses. Sufficient information and source data have been provided to allow others to repeat every step of the work Yes, I think there is sufficient information to repeat the work. The conclusions are supported by the findings. No, I think that there are a few things that need to be addressed in the discussion. First, please refer to the references I listed above and the articles that have cited them since, and re-think the discussion in that new framework. Second, the statistical issues of having all of 1 treatment in 1 row need to be addressed, otherwise all statistical results could be considered related to some unknown environmental gradient. Some minor comments: The introduction is mostly about community assembly. However, to me, the really interesting thing about this study is that it is done using a major agricultural crop. Little is discussed about why it would be interesting to know about bacterial community assembly in this plant. Perhaps there is a context for plant productivity or agricultural disease that could be expanded to make the intro more specific to the study? We added more text to the first paragraph of the introduction to incorporate agricultural context. We are pleased that you find this interesting! There is currently substantial investment in the business sector around plant microbiomes and probiotics (several hundred million dollars, and at least four new companies that we know of), but there is relatively little publicly available basic research on agricultural plant microbiomes. We added a citation from one review of this area (Parnell et al., 2016). I found the organization of the manuscript difficult to follow. Please reorder so that all SEM-related sections are together and all ARISA sections are together in the methods. Then follow whatever order you have chosen for the results. We moved the SEM image analysis section to the correct place after the SEM image acquisition section in the methods. Thank you for pointing this out! The supplementary data tables are simply raw R output. Can you summarize the raw output in a supplementary table and then provide the raw output so that it is easier for the reader to interpret? These are output from the Primer-E program with full details of the model fits and outcomes. We added more explanation to the methods for Tables 1 to 4 to make this clear. It would be easier to interpret Tables 1 and 2 if the significances were just included in Figure 3 instead of in a separate table. (i.e. put the letters above each of the averages shown in Figure 3 to indicate significances) The permdisp/permanova and non-parametric richness analyses have different assumptions, so we chose not to place them in the same figure. Figure 3 - the red/green dots are difficult to see (and I am not red/green color blind, so it would be even harder for 10% of our colleagues). Please adjust these so that they are more obviously different or have different shapes. We changed these to different colors (manually). We agree that these are not correctly formatted and we have submitted a request to the company that developed the program asking them to adjust their defaults to color-blind-compatible colors and shapes. Supplementary Figure 3 needs a legend that identifies what each of the SCU_IDs are and more information about the graph. We have added the caption to this figure. On page 9, the authors state that \"In the ARISA datatype, highly variable co-occurring…\". Please provide more of a definition of what you mean by \"highly variable\". Is this quantifiable? We added more explanation to this sentence. Since network interaction patterns are a summary measure (and thus not replicated) we do not discuss them quantitatively. Discussion Section - there are 3 different referencing styles used. Change for consistency. Updated to the F1000 style. Is the work clearly and accurately presented and does it cite the current literature? No Is the study design appropriate and is the work technically sound? Partly Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? No Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly References 1. Redford AJ, Bowers RM, Knight R, Linhart Y, Fierer N: The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves.Environ Microbiol. 2010; 12 (11): 2885-93 PubMed Abstract \| Publisher Full Text 2. Kembel SW, O'Connor TK, Arnold HK, Hubbell SP, Wright SJ, Green JL: Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest.Proc Natl Acad Sci U S A. 2014; 111 (38): 13715-20 PubMed Abstract \| Publisher Full Text Competing Interests: No competing interests were disclosed. Referee Expertise: Microbial ecology, soil, airborne, biodegradation I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above." } ] } ]	1	https://f1000research.com/articles/6-1698
https://f1000research.com/articles/6-1415/v1	10 Aug 17	{ "type": "Research Article", "title": "Diagnosis of three different pathogenic microorganisms by gas chromatography-mass spectrometry", "authors": [ "Najmeh Karami", "Fateme Mirzajani", "Hassan Rezadoost", "Abdollah Karimi", "Fatemeh Fallah", "Alireza Ghassempour", "Atusa Aliahmadi", "Najmeh Karami", "Fateme Mirzajani", "Hassan Rezadoost", "Abdollah Karimi", "Fatemeh Fallah", "Alireza Ghassempour" ], "abstract": "Background: Diagnoses of respiratory tract infections usually happen in the late phase of the disease and usually result in reduction of the pathogen load after broad-spectrum antibiotic therapy, but not in eradication of the pathogen. The development of a non-invasive, fast, and accurate method to detect pathogens has always been of interest to researchers and clinicians alike. Previous studies have shown that bacteria produce organic gases. The current study aimed to identify the volatile organic compounds (VOCs) produced by three respiratory tract pathogens, including Staphylococcus aureus, Escherichia coli and Candida albicans. Methods: The volatile organic compounds (VOCs) produced were identified by gas chromatography–mass spectrometry(GC-MS), with prior collection of microbial volatile compounds using solid phase microextraction (SPME) fiber. The volatile compounds were collected by obtaining bacterial headspace samples. Results: Results showed that these three organisms have various VOCs, which were analyzed under different conditions. By ignoring common VOCs, some species-specific VOCs could be detected. The most important VOC of E. coli was Indole, also some important VOCs produced by S. aureus were 2,3-Pentandione, cis-Dihydro-α-terpinyl acetate, 1-Decyne, 1,3-Heptadiene-3-yne, 2,5-dimethyl Pyrazine, Ethyl butanoate and Cyclohexene,4-ethenyl furthermore, most of identified compounds by C. albicans are alcohols. Conclusions: The detection of VOCs produced by infectious agents maybe the key to make\n\na rapid and precise diagnosis of infection, but more comprehensive studies must be conducted in this regard.", "keywords": [ "Candida albicans", "Escherichia coli", "gas chromatography-mass spectrometry", "Staphylococcus aureus", "volatile organic compounds" ], "content": "Introduction\n\nInfectious diseases are the main reason for morbidity and mortality in developing countries, especially among children1. Staphylococcus aureus is a common inhabitant of the upper respiratory tract in children, and the causative agent for many infections. It is believed that people under 20 are more likely to have these bacteria. There is a greater possibility that S. aureus exists in the respiratory tract of infants aged 3 months or younger than in people of other ages2. Moreover, S. aureus is colonized in the nasopharynx in 10–35% of children, and in almost 35% of the adult population3.\n\nEscherichia coli is one of the most significant pathogens affecting preterm infants4. Some studies in developing countries have suggested that gram-negative rods (such as E. coli) are the major causes of infection in premature infants (0–6 days)5–7. Furthermore, infections caused by E. coli are one of the most important causes of death in the early neonatal period5. Candida albicans is an opportunistic pathogen and an agent of nosocomial infection8.\n\nGenerally, the causative agents of respiratory tract infections are diagnosed in late phases of the disease7. Such infections need broad-spectrum antibiotic therapy, the consequences of which are a reduction in the pathogen load, but not eradication. Moreover, such therapies increase the probability of drug-resistant infections spreading9. Accurate and rapid detection of pathogens is a critical step for adequate treatment of infection10. and a non-invasive diagnostic method that has a high degree of accuracy needs to be developed11.\n\nIt has been shown that bacteria produce organic gases. Different types of microorganisms have a distinct metabolism, and they produce various types of volatile organic compounds (VOCs)12–14. Attempts have been made to identify the VOCs of pathogenic organisms15–20. There are several sophisticated methods available that have been used for recognizing VOCs; these include gas chromatography-mass spectrometry (GC-MS)21, selected ion flow tube mass spectrometry (SIFT-MS)22, electronic noses (eNoses)23, and ion-molecule reaction mass spectrometry (IMRMS)24. Previous studies suggest that GC-MS is the most appropriate and reliable technique for the isolation and identification of VOCs25–27.\n\nThe current study aimed to identify the volatile organic compounds (VOCs) produced by three respiratory tract pathogens, including Staphylococcus aureus, Escherichia coli and Candida albicans, to determine if these could be used as biomarkers.\n\n\nMaterials and methods\n\nThe bacterial strains used in this study were E. coli (ATCC 25922) and S. aureus (ATCC 25923), as gram-negative and gram-positive model organisms, and C. albicans (ATCC 10231) was used as a human pathogenic fungi model. These organisms model were obtained from the Microbiology Laboratory of Medicinal plants and Drugs Research Institute, Shahid Beheshti University. Monocultures of all strains were cultured 24 hours in nutrient agar, and then sub-cultured aerobically at 37°C in 30 ml of two different types of broth medium, Mueller Hinton broth (MB) and tryptic soy broth (TSB), in 100 ml sterilized glass bottles. For a more careful assessment of VOCs produced by each microorganism, the headspace was extracted from both media at three different time points:2, 4 and 24 hours. To increase the possibility of VOC production, bottles containing cultured microorganism were shaken at 150 rpm during incubation time28. A suspension of microorganisms with OD600 ~0.5 in culture media was used during the headspace extraction10, and the corresponding sterile broth mediums were used as the blank samples29.\n\nA solid phase microextraction (SPME) fiber holder (57330-U, Sigma-Aldrich) containing fiber coated with divinyl benzene/carboxen/poly dimethyl siloxane 50/30 µm (DVB/CAR/PDMS) (57328-U, Sigma-Aldrich) was used for absorption of volatile compounds from the headspace of pathogens. To provide conditions that increase the rate of VOC absorption, after incubation time, 2ml of NaCl 36% was added to each culture. Then the DVB/CAR/PDMS fiber was suspended from the top of the bottle containing the culture and placed on a magnetic stirrer hotplate at 70°C for 30 minutes30. After that, the fiber was placed at the injection site of GC-MS and all the absorbed VOCs entered the device. Eventually each VOC is represented as a chromatogram peak in the monitor that is connected to the GC-MS. For thermal desorption, the SPME fiber remained in the injector for 2 minutes before it was exposed to the headspace of the pathogen samples31.\n\nTo study the bacterial VOCs, a Thermo-Finnigan Trace GC-MS system (Thermo Quest-Finnigan Co) equipped with a DB-5 column (60 m length, 0.25 mm inner diameter, and 0.25 μm film thickness) with helium carrier gas at a flow rate of 1.1 ml/min was used. The starting temperature was 50°C, increasing at a rate of 10°C/minute up to 250°C. The GC-MS was set in splitless mode and a quadrupole ion trap with ionization energy of 70 eV was used in the filament.\n\nVOCs were identified using the National Institute of Standards and Technology (NIST) reference library. To analyze the GC-MS data, Xcalibur 3.0 with Foundation 3.0 SP2 software (Thermo Fisher Scientific) was used, and the kovats retention index (RI) was calculated for each chromatographic peak.\n\nWhen calculating the RI, a series of standards were used: n-alkanes were injected into the GC-MS the day before starting experiments, using the same temperature profile that would be used for the analysis of VOCs. The NIST17 Mass Spectral Library (NIST7/2017/EPA/NIH) was used to identify each compound according to its RI. Extensive studies were also performed by a phytochemist to determine if the compounds were organic.\n\n\nResults\n\nThe VOCs produced by S. aureus, E. coli and C. albicans were assessed under six different conditions (using two types of media and taking measurements at three time points). The Xcalibur raw files for these three pathogens are available at https://doi.org/10.6084/m9.figshare.5178004.v132.\n\nOne chromatogram of the six chromatograms obtained is displayed in Figure 1, showing the chromatogram obtained 4 hours after culture in TSB medium, for each pathogen. The five other chromatograms are also available, as Supplementary File S1, Supplementary File S2, Supplementary File S3, Supplementary File S4 and Supplementary File S5.\n\nThe other chromatograms are available in the Supplementary material.\n\nThe processed GC-MS data obtained in the current study is available in a total of 18 tables as supplementary GC-MS data. It shows the details of the VOCs detected for each of the three pathogens, each analyzed under different conditions (using two types of media and taking measurements at three time points, as explained above).\n\nFor a better overview the detected VOCs are shown in three tables (at the 2 hour time point in Table 1, at the 4 hour time point in Table 2 and at the 24 hour time point in Table 3), alongside the percentage of the total area that the average peak of the detected VOC covered. In other words it is proportional to amount of the compound that is present.\n\nIn total, 26 types of VOCs by E. coli, 34 types by S. aureus and 29 types by C. albicans were generated in this period.\n\nIn total, 9 types of VOCs by E. coli, 19 types by S. aureus and 43 types by C. albicans were generated in this period.\n\nIn total, 16 types of VOCs by E. coli, 26 types by S. aureus and 28 types by C. albicans were generated in this period.\n\nSome VOCs were common among organisms and were generated by two or three organisms at an approximately equal rate, including 1,2-Benzene dicarboxulic acid, 1,9-Decadiene, 2,5-(1,1-dimethylethyl)-phenol, 2,6-bis(1,1-dimethylethyl)-4-methyl-phenol, 3-Propionyl oxy pentadecane, Anisol and Dibutyl phatalate (Table 1). Some common VOCs were produced at a greater rate between one organism and another. It can be concluded that these VOCs could also be more important in the organism that produces greater quantities. 1-Penten-3-ol was produced from E. coli in TSB medium after 2 hours (0.02%); under identical conditions, more of it was produced by S. aureus (5.14%) than by E. coli. Furthermore, Indole was produced from E. coli after 2 hours of culture in two types of medium (82.61% for MB and 90.97% for TSB) and was also produced by S. aureus after 2 hours in TSB medium, although at a much lower rate (0.48%) (Table 1).\n\nUncommon VOCs of E. coli detected 2 hours after culture included 1-(1,5-dimethyl)-4-hexyl-4-methyl-Benzene, 2,3-Pentandione, 2,6-dibutyl-2,5-cyclohexadiene-1,4-dione, Benzophenone, Bisabolene, Copaene, Decanol, Dodecanol, Indole, Limonene, Muurola-4,5-diene, Neryl acetate, Phenyl ethyl Pyrrole, Sesquiphellandrene and Tetradecane (Table 1).\n\nUncommon VOCs of S. aureus detected 2 hours after culture included 1,2-Butadiyene, 1-Penten-3-ol, 2,5-dimethyl Pyrazine, 2-Ethyl hexanol, Allyl butyl hydroquinone, Benzene acetaldehyde, Butyl cyclohexyl acetate, Caryophyllene, Cedrol, Cyclohexene, 4-ethenyl-, Decene, Dimethyl Octenal, Heptadecane, Humulen, Longifolrne, Methone, Nonadecanone and Tetrabutyl cyclohexyl acetate (Table 1).\n\nUncommon VOCs of C. albicans detected 2 hours after culture included 1,3-Butadiyene, 1,5-Decadiene, 2-Hexan-1-ol, 3-Methyl-1,5-heptadiene, butyraldehyde, Cadinene, Carbamic acid, Dodecenol, Eicosane, Ethyl butanoate, Longifolene, Ocimene, Octyl acetate, Tetradecanol, β-Santaloland β-Sesquiphellandrene (Table 1).\n\nUncommon VOCs of E. coli identified 4 hours after culture included 1,9-Decadiene, 2-Acetyl-1-pyrroline, 2-Heptanone and Indole (Table 2).\n\nUncommon VOCs of S . aureus identified 4 hours after culture included 1,2-Butadiyene, 1,3-Heptadiene-3-yne, 1-Decyne, 1-Methoxy-2-propanol, 2,3-Pentandione, 2-Ethyl hexanol, 2-methyl-2-Undecanethiol, Butyraldehyde, cis-Dihydro-α-terpinyl acetate, Cyclohexene,4-ethenyl- and Levomenthol (Table 2).\n\nUncommon VOCs of C. albicans identified 4 hours after culture included (E)-2-hexyl ester- Butanoic acid, (z)-2-Octene-1-ol, (z)-4-Decan-1-ol, 1,2-Benzenedicarboxulic acid, 1,3-Butadiyene, 1,5-Decadiene, 2-(phenyl methylene)-Octanal, 2,5-(1,1-dimethylethyl)-Phenol, 2,5-dimethyl Pyrazine, 2-ethenyl-6-methyl-Pyrazine, 2-Hexan-1-ol, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, 5.5-Dodecadinyl-1, 12-diol, 6-Methyl-5-hepten-2-one, Benzaldehyde, Cadinene, Carbamic acid, Cedrol, Dibutyl phatalate, Dimethyl Octenal, Dimethyl ethyl Cyclohexanol, Dodecenal, Dodecenol, Eicosane, Longifolene, Longifolol, Methyl isopropyl Hexenal, Naphthalenol, Octacosane, Octyl acetate, Phatalic acid butyl ester, Tetradecanol, Tridecanol, Zingiberene and β-Sesquiphellandrene (Table 2).\n\nUncommon VOCs of E. coli identified 24 hours after culture included 1,9-Decadiene, 2-Acetyl-1-pyrroline, 2-Heptanone, 2-Methyl tetradecane and Indole (Table 3).\n\nUncommon VOCs of S. aureus identified in 24 hours after culture were included; 1,2-Butadiyene, 1,3-Heptadiene-3-yne, 1-Decyne, 1-Methoxy-2-propanol, 2,3-Pentandione, 2,5-Dimethyl pyrazine, 2-Decenal, 2H-Tetrazole-5-carboxylicacid, 2-phenyl, 3-Methyl-1,5-heptadiene, Caryophyllene, cis-Dihydro-α-terpinyl acetate, Cyclohexene,4-ethenyl-, Ethyl butanoate, Levomenthol and Thiophene (Table 3).\n\nUncommon VOCs of C. albicans identified 24 hours after culture included (z)-2-Octene-1-ol, (z)-4-Decan-1-ol, 1,2-Benzene dicarboxulic acid, 1,5-Decadiene, 2-(phenyl methylene)-Octanal, 2,5-dimethyl Pyrazine, 2-methyl-1-propanol, 2-methyl-2-Undecanethiol, 2-octyl-1-ol, 2-octyne, 3-Methy-4-pentene-3-ol, 3-methyl-1-pentene, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, Carbamic acid, Cedrol, Dibutyl phatalate, Ethyl acetoacetate, Longifolol, Octacosane and Zingiberene (Table 3).\n\n\nDiscussion\n\nAs previous studies have shown, organisms are able to produce either common or specific VOCs33–35. In the current study, GC-MS was used to detect VOCs generated by three pathogenic organisms in the human respiratory tract. The VOCs of E. coli, S. aureus and C. albicans were analyzed at three different time points, using two different types of media (Figure 1).\n\nResults of the current study suggest that VOCs exclusively produced by E. coli are 1-(1,5-dimethyl)-4-hexyl-4-methyl-Benzene, 2,6-dibutyl-2,5-cyclohexadiene-1,4-dione, Benzophenone, Bisabolene, Copaene, Decanol, Dodecanol, Indole, Limonene, Muurola-4,5-diene, Nerylacetate, Phenyl ethyl Pyrrole, Sesquiphellandrene, Tetradecane, 2-Acetyl-1-pyrroline and 2-Methyl tetradecane. The most important compound among these is Indole, because it is generated at the three time points and also it was the most produced VOC by E. coli (at least 82%). Other studies have confirmed this finding28,29,35.\n\nThe current study has shown that the specific VOCs produced by S. aureus are 1,2-Butadiyene, 1-Penten-3-ol, 2,5-dimethyl Pyrazine, 2-Ethyl hexanol, Allyl butyl hydroquinone, Benzene acetaldehyde, Butylcyclohexyl acetate, Caryophyllene, Cyclohexene, 4-ethenyl-, Decene, Heptadecane, Humulen, Longifolrne, Methone, Nonadecanone, Tetrabutylcyclohexyl acetate, 1,3-Heptadiene-3-yne, 1-Decyne, 1-Methoxy-2-propanol, 2,3-Pentandione, cis-Dihydro-α-terpinyl acetate, Levomenthol, 2-Decenal, Ethyl butanoate and Thiophene. Moreover, 1,2-Butadiyen, 2,5-dimethyl Pyrazine, 2-Ethyl hexanol, Caryophyllene, Cyclohexene, 4-ethenyl, 1,3-Heptadiene-3-yne, 1-Decyne, 1-Methoxy-2-propanol, 2,3-Pentandione, cis-Dihydro-α-terpinyl acetate, and Levomenthol. They were detected under more than one of the six conditions that were tested, so they are significant. Another important point is that the percentage of the total area that the average peaks for 2,3-Pentandione, cis-Dihydro-α-terpinyl acetate, 1-Decyne, 1,3-Heptadiene-3-yne, 2,5-dimethyl Pyrazine, Ethyl butanoate and Cyclohexene,4-ethenyl covered were at least 15%; thus, they are remarkable VOCs for S. aureus. Some of the VOCs produced by S. aureus in the current study have been reported in other studies34,36 but some of them have not11,33.\n\nThis study suggested that the specific VOCs produced by C. albicansinclude 1,3-Butadiyene, 1,5-Decadiene, 2-Hexan-1-ol, Cadinene, Carbamic acid, Dodecenol, Eicosane, Longifolene, Ocimene, Octyl acetate, Tetradecanol, β-Sesquiphellandrene, (z)-2-Octene-1-ol, (z)-4-Decan-1-ol, 2-(phenyl methylene)-Octanal, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, Longifolol, 6-Methyl-5-hepten-2-one, Dodecenal, Methyl isopropyl Hexenal, Tridecanol, 2-methyl-2-Undecanethiol, 2-octyl-1-ol, 2-octyne, 3-Methy-4-pentene-3-ol, 2-methyl-1-propanol and 3-methyl-1-pentene. Also, 1,3-Butadiyene, 1,5-Decadiene, 2-Hexan-1-ol, Cadinene, Carbamic acid, Dodecenol, Eicosane, Longifolene, Octyl acetate, Tetradecanol, β-Sesquiphellandrene, (z)-2-Octene-1-ol, (z)-4-Decan-1-ol, 2-(phenyl methylene)-Octanal, 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane, Longifolol, Octyl acetate, β-Sesquiphellandreneand 2-methyl-2-Undecanethiol were detected under more than one of the six conditions that were tested, so they are significant. Furthermore, 1,3-Butadiyene, Carbamic acid, Longifolol, β-Santalol, 2-methyl-1-propanol, 2-methyl-2-Undecanethiol and 4-t-butyl-2-(1-methyl-2-nitroethyl) cyclohexane were produced in greater quantities. Several studies have analyzed the VOCs of C. albicans and have noted that most of these identified compounds are alcohols37–39.\n\nFinding a non-invasive and rapid method for diagnosis of infectious agents is a subject of interest, so it has been investigated in several studies33,40–43. The current study showed that using SPME fiber and GC-MS for extraction and detection of VOCs allowed detection of more specific VOCs for the three pathogenic respiratory tract organisms, E. coli, S. aureus and C. albicans, which could be used as biomarkers for their identification. It is essential that more comprehensive studies be conducted to create a more complete profile of VOCs for these organisms, and so that the methods can be developed further.\n\n\nData availability\n\nThe Xcalibur raw files for the three studied pathogens are available at https://doi.org/10.6084/m9.figshare.5178004.v132.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Shahid Beheshti University of Medical Sciences.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThis paper is part of Najmeh Karami’s PhD thesis, named: \"Identification the volatile metabolic profiling of six respiratory pathogenic organisms\". We would like to express our specific thanks to Shahid Beheshti University of Medical Sciences for the financial support. We also appreciate all colleagues that helped us in the Pediatric Infections Research Center, Mofid Children’s Hospital, Shahid Beheshti University of Medical Sciences and Medicinal Plant and Drug Research Institute, Shahid Beheshti.\n\n\nSupplementary material\n\nSupplementary File S1: Three chromatograms, for samples taken 2 hours after culture in MB media. EC: E. coli, SA: S. aureus and CA: C. albicans.\n\nClick here to access the data.\n\nSupplementary File S2: Three chromatograms, for samples taken 4 hours after culture in MB media. EC: E. coli, SA: S. aureus and CA: C. albicans.\n\nClick here to access the data.\n\nSupplementary File S3: Three chromatograms, for samples taken 24 hours after culture in MB media. EC: E. coli, SA: S. aureus and CA: C. albicans.\n\nClick here to access the data.\n\nSupplementary File S4: Three chromatograms, for samples taken 2 hours after culture in TSB media. EC: E. coli, SA: S. aureus and CA: C. albicans.\n\nClick here to access the data.\n\nSupplementary File S5: Three chromatograms, for samples taken 24 hours after culture in TSB media. EC: E. coli, SA: S. aureus and CA: C. albicans.\n\nClick here to access the data.\n\nSupplementary File S6: GC-MS data analysis, showing the details of the detected VOCs of three pathogens in 6 modes.\n\nClick here to access the data.\n\n\nReferences\n\nRodríguez L, Cervantes E, Ortiz R: Malnutrition and gastrointestinal and respiratory infections in children: a public health problem. Int J Environ Res Public Health. 2011; 8(4): 1174–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang YC, Chen CJ: Nasal carriage of methicillin-resistant Staphylococcus aureus during the first 2 years of life in children in northern Taiwan. Pediatr Infect Dis J. 2015; 34(2): 131–5. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "25394", "date": "08 Sep 2017", "name": "Paul Brinkman", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGeneral Comments:\nThis study aimed to identify typical volatile organic compounds (VOCs) produced by Staphylococcus aureus, Escherichia coli and Candida albicans by extraction of cultured bacterial strain headspace using solid phase microextraction (SPME) fibers analyzed by Gas Chromatography-Mass Spectrometry (GC-MS). Although the aim of study is obvious and very relevant data is shown, some extension on utilized statistics is needed. Additionally, a replication and/or validation of findings is crucial. The authors do show that some of the VOCs, e.g. Indole, are revealed at multiple time points, but confirmation of observations by repetition of experiments is recommended.\n\nMajor Comments:\nTo avoid possible false discoveries a repetition of experiments is recommended.\n\nWhich (statistical) methods were used in order to compare blank vs. actual samples?\n\nWhich criteria were applied in order to confirm a ‘match’ between detected volatiles and the NIST library?\n\nThe authors nicely show the overlap and/or discrepancy between their findings and current available literature, but what about the interpretation of findings? Can the authors extend and/or speculate about possible biological mechanisms behind the revealed VOCs\n\nMinor comment:\n\nPlease consider as reference: Neerincx et al; Identification of Pseudomonas aeruginosa and Aspergillus fumigatus mono- and co-cultures based on volatile biomarker combinations; J Breath Res. 2016 Jan 29;10(1):016002.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "25909", "date": "15 Nov 2017", "name": "Amy Scott-Thomas", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper looks at the detection of VOC's produced by Escherischia coli, Staphylococcus aureus and Candida albicans using solid phase micro-extraction coupled with gas chromatography / mass spectrometry.\n\nIts seems as though the group only ran this experiment once so while they have pointed out the detection of specific volatiles at differing time points this needs to be repeated to ensure that it is indeed a true and repeatable release from the micro-organisms.\nThe work is outlined clearly and the results listed comprehensively however there has been no indepth discussion surrounding why these volatiles have been produced in vitro, would they be produced in vivo nor an extensive list of references to back this up.\n\nWhile they state Indole as the most important volatile produced by E.coli they do not go into any detail regarding how this would relate to an actual breath test since indole can be released by mouth flora. Also as indole is produced from tryptophan discussion around the levels of tryptophan in the lung and therefore its actual availability to E.coli growing in the lung is pertinent. Growth in a minimal media with varying tryptophan levels would give more insight.\nIt would have also been beneficial to look at the effect of co-culturing these micro-organisms as this may alter the release of the detected VOC's and initiated the production of others.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "27814", "date": "27 Nov 2017", "name": "Norman Ratcliffe", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe researchers tackle an area of significant interest, rapid determination of bacterial species, especially associated with life threatening illness, using a relatively new approach, that of VOC analyses. In general the paper reads well.\nThe analytical method for VOC analyses is ok. Could the authors confirm whether the cfu/ml count is approx. for the different species at the same time points to enable a good comparison of VOCs to be made. One major weakness to the work is that only one analysis for each species at one time point and media was undertaken, the literature gives examples of several analyses being undertaken for similar studies. The title is too ambitious, rather than diagnoses being purported, maybe an” initial study of…” would be more accurate.\nThe authors could comment on how their research would ultimately fit into a clinical test, if the VOCs were to be analysed in breath, would the same volatile profile be expected?\nIn the text it was stated that “Extensive studies were also performed by a phytochemist to determine if the compounds were organic” , some explanation would be good as to what this means.\nSome other matters, VOC abbreviation used twice in the abstract, also in quite a few places the chemical names have capital letters and are misspelt and merged with another word.\nIn Results, what is uncommon determined by, some more discussion on stats…\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? No\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-1415
https://f1000research.com/articles/7-74/v1	17 Jan 18	{ "type": "Opinion Article", "title": "How to prevent the next Marathon Pharmaceuticals", "authors": [ "Frank S. David", "Richa Dixit", "Richa Dixit" ], "abstract": "In recent years, several drug companies have exploited U.S. regulatory policies to acquire exclusive rights to cheap therapies and substantially raise their prices, and Federal agencies and state governments are exploring various ways to prevent or punish such behavior in the future. Among these cases, however, Marathon Pharmaceuticals’ handling of Emflaza (deflazacort) is unique, because the drug was previously only available abroad, and was never previously sold in the U.S. before the company obtained FDA approval for it. Thus, laws and policies designed to address price hikes on already-marketed drugs are unlikely to prevent additional Marathon-like scenarios. In this article, we describe in more detail the unique features of Emflaza compared with these other recent cases of drug price increases, determine the likelihood that similar situations will arise in the future, and explore legislative and administrative options to specifically prevent such behavior.", "keywords": [ "FDA", "orphan", "pharmaceutical", "price", "regulation" ], "content": "Introduction\n\nWhen Marathon Pharmaceuticals obtained U.S. approval for Emflaza (deflazacort) in Duchenne’s muscular dystrophy (DMD) in 2017, legislators and industry critics vilified the company’s plan to price the drug at $89,000 per year – not for the high cost per se, but for allegedly taking unfair advantage of U.S. regulatory and pricing policies to profit handsomely from a decades-old drug available abroad for under $5 a day.\n\nAlthough Marathon gained the public’s attention in the wake of several other high-profile cases of alleged drug pricing misconduct, this case is actually unique. Unlike firms that have acquired U.S.-marketed agents and then hiked their prices, Marathon gained FDA approval of a drug that was already commercialized abroad, but not in the U.S., then set a U.S. price far higher than the international one. This distinction does not automatically exonerate Marathon, but it highlights a special challenge for regulators and policy-makers seeking to balance access with affordability. It also suggests that there may not be a “one size fits all” approach to punishing and/or preventing companies that engage in the broad range of improper drug pricing behaviors that have been seen to date.\n\nThe details of the Marathon case raise three questions. First, what specific aspects of the company’s behavior were inappropriate, and distinguish Marathon from other companies that launch high-priced drugs in the U.S.? Second, how prevalent and important are future scenarios like Emflaza likely to be? And finally, what legislative, administrative, and non-policy approaches might effectively prevent and/or punish similar activities?\n\n\nDistinguishing Emflaza from “appropriate” orphan drugs\n\nDeflazacort, a synthetic corticosteroid originally developed by Merrell Dow Pharmaceuticals, has been available since the 1990s outside the U.S. In 1996, preliminary data in 196 DMD patients showed it was comparably effective to prednisone, but had fewer side effects.1 Since then, physicians outside the U.S. have prescribed it off-label for DMD, and some American patients have reportedly obtained it from abroad, as permitted under FDA’s personal importation policies.a Marathon Pharmaceuticals purchased the rights to deflazacort, completed the data analysis2, and received FDA approval for the drug (now called Emflaza) in February 2017.b\n\nMarathon’s announcement that it intended to raise Emflaza’s U.S. price 60-fold compared with its price in the U.K. attracted substantial scrutiny and censure. Experts estimate Marathon spent about $50M, and possibly far less, to acquire deflazacort and conduct limited additional research. In return for that relatively meager investment, the company was rewarded with seven years of market exclusivity under the Orphan Drug Act for a drug that could earn over $400M annually.\n\nSuperficially, Emflaza invites comparison with other recent examples of exorbitant drug price increases taken by companies that invested little to nothing in the therapy’s development. For example, in 2013, Marathon itself acquired two FDA-approved cardiac therapies, Nitropress (sodium nitroprusside) and Isuprel (isoproterenol), from Hospira, then increased the prices about 4-fold before selling the drugs to Valeant in 2015, which immediately further hiked the prices. And in 2015, Turing Pharmaceuticals acquired Daraprim (pyrimethamine), a drug for toxoplasmosis and other infectious diseases, and within months raised the price over 50-fold. In these cases, like with Emflaza, a company reaped outsized returns by substantially increasing the price on a marketed drug that was discovered long ago by another firm.\n\nBut Marathon is distinct from these other examples of egregious price increases because before the company gained approval for Emflaza, U.S. physicians could not prescribe deflazacort to DMD patients. By submitting previously unpublished data on the drug for FDA regulatory review, Marathon enabled Americans to easily obtain a clinically useful therapy that was previously inaccessible through normal channels. This is in stark contrast to the examples cited above, as well as other recent cases of excessive price hikes, like cycloserine, Duexis (ibuprofen and famotidine)3, Keveyis (daranide), and Vimovo (esomeprazole and naproxen) – because in all of these other cases, American patients already had unfettered access to the drug (or its active ingredients).\n\nThe fact that Emflaza was not previously available by prescription in the U.S. does not itself justify Marathon’s pricing decision, but it highlights a distinctive aspect of this case compared with the other examples cited above. Marathon certainly benefitted from pricing freedom and regulatory advantages that are intended for novel drugs that require substantial at-risk R&D investment, which was not the case with Emflaza. At the same time, however, there is value to enabling American patients to access deflazacort, an effective drug in an under-served disease area, through a standard prescription, without the hassle and expense of acquiring it from abroad. Emflaza illustrates the tension between protecting Americans from predatory pricing behavior on the part of drug manufacturers and encouraging companies to launch meaningful therapies in the U.S. Thus, this particular scenario is not a clear-cut, routine example of pharmaceutical price-gouging, and may warrant specific attention from legislators and regulators.\n\n\nDefining the magnitude of the problem\n\nBefore considering ways to balance affordability and access in Emflaza-like scenarios, it is worth understanding how common these situations are likely to be in the future. To answer this question, we searched for other “legacy” drugs like deflazacort that are available abroad and have existing off-label data in an orphan indication that might support speedy FDA approval at low cost and risk.c We focused on orphan drug opportunities because these are most likely to be attractive to biotechs seeking to pursue this strategy: published data from a small trial may suffice for FDA approval, without the need for additional studies; high prices for rare diseases therapies in the U.S. are common, and rarely encounter backlash from payers; and market exclusivity under the Orphan Drug Act makes this strategy feasible for “legacy” therapies.\n\nUsing the U.K. as a test case, we identified just one approved drug that we believe could support an Emflaza-like strategy: celiprolol, an off-patent beta-blocker originally developed by Rhône-Poulenc and available cheaply abroad. Published data show that celiprolol prophylaxis significantly reduces arterial rupture and dissection in vascular Ehlers-Danlos syndrome (vEDS), a genetic collagen disorder.4 Thus, we found it highly possible that a firm could obtain the rights to the drug and data, garner FDA approval with little or no additional R&D expense, raise the price substantially over its current U.K. price, and gain orphan drug market exclusivity. In fact, during the preparation of this article we found that a biotech firm, Acer Therapeutics, had already obtained exclusive rights to celiprolol’s vEDS data, and intends to file for FDA approval for the drug (to be called Edsivo) in early 2018, having disclosed no plans for additional clinical trials. Although we analyzed just one non-U.S. country for illustrative purposes, we believe our results suggest that future scenarios directly analogous to Emflaza are possible, but unlikely to be frequent.\n\n\nPreventing (or punishing) the next Marathon\n\nEmflaza represents a special case of the general problem of companies taking egregious price increases on old drugs, because it involves a therapy not previously approved by the FDA. Thus, recent state and Federal activities aimed at curbing high prices on off-patent drugs that lack generic competitors or large price increases on already-approved agents will not prevent or punish future Emflaza-like scenarios.\n\nThe most effective and least burdensome approach to prevent other firms from copying Marathon’s Emflaza strategy would likely involve alterations to FDA’s existing rules on personal importation.d Americans are currently permitted to import drugs for non-serious conditions for personal use if they are “not known to represent a significant health risk”. For “serious” conditions, importation of drugs deemed safe is allowed for personal use only if the drug is neither available nor promoted commercially in the U.S. Refining these criteria to allow Americans to import generics in certain situations where a drug with the same active ingredient is already available in the U.S. – for example, if the generic was launched abroad before the first FDA approval – would permit continued importation of drugs like deflazacort and celiprolol, and thus eliminate the incentive for future companies to take a similar approach. At the same time, such a change would not jeopardize the incentives for drug developers to invest in R&D to develop bona fide new therapies and bring them to the U.S. market.\n\nOther options to prevent or punish firms pursuing Marathon’s Emflaza strategy are also theoretically possible, but may be more challenging to implement. It is extremely difficult to define criteria that could allow FDA to block drugs like Emflaza while still approving “legitimate” drugs applying for U.S. market access (based, for example, on an (arbitrary) minimum amount of R&D investment), and in the absence of such a definition, providing FDA with broad latitude to refuse approval to drugs like Emflaza on a case-by-case basis – a sort of “I know it when I see it” approach to drug price gamesmanship – is unlikely to withstand legal scrutiny. FDA could also enact policies to automatically grant FDA approval to drugs already available outside the U.S., but versions of such “reciprocal approval” policies proposed to date could have undesirable safety implications for American patients that have not been fully explored.5 And on the pricing side, although there has been significant recent effort in state legislatures to control price increases on drugs already available in the U.S., there has been scant progress on fairly controlling launch prices of new drugs, which is the key issue for Emflaza.6\n\n\nConclusions\n\nAlthough pharmaceutical industry critics commonly lump together various drug price scandals, there are important differences between them that may require, in some cases, bespoke solutions. Marathon Pharmaceuticals illustrates a very specific challenge for legislators and regulators: the potential for companies to gain FDA approval for old drugs available outside the U.S., often under policies intended to support innovative R&D in rare diseases, and then take advantage of the permissive American drug pricing environment to substantially raise their prices. This situation, though likely to be rare in the future, will not be prevented by most of the drug pricing policies currently under consideration at the state and Federal level, and thus may warrant focused attention and a customized approach. Among several potential solutions, a change to FDA’s personal importation policies is most likely to prevent future Emflaza-like scenarios while having little to no impact on the broader U.S. system that rewards drug companies for investing in risky, expensive R&D in clinically important new therapies.", "appendix": "Competing interests\n\n\n\nThe authors work for Pharmagellan, a pharmaceutical consulting firm. Neither author has ever been employed as a consultant to any of the companies named in this article, nor does either author have any current or prior investments in any of the named companies.\n\n\nGrant information\n\nThe author(s) declare that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Frank Provenzano for assisting with initial data capture and analysis, and Julie Lin, Mark Lindsay, Robert Nussbaum, Lisa Rosenbaum, and Rachel Sachs for providing helpful comments during the preparation of this manuscript. We also thank two anonymous reviewers at Annals of Internal Medicine, whose helpful suggestions led us to make substantial improvements to this article.\n\n\nSupplementary material\n\nSupplementary File 1: Potential future “Emflaza-like” opportunities subjected to further analysis. Active ingredients available by prescription in the U.K., but not the U.S., on February 18, 2017, that we analyzed manually (N=133). See text for details.\n\nClick here to access the data.\n\n\nFootnotes\n\na U.S. FDA Regulatory Procedures Manual, Ch. 9: Import Operations and Actions.\n\nb The company subsequently sold the rights to another biotech company, PTC Therapeutics.\n\nc On February 18, 2017, we downloaded complete lists of drugs approved in the U.S. (2,610) and U.K. (1,879) from https://www.fda.gov/Drugs/InformationOnDrugs/ucm079750.htm and https://www.medicines.org.uk/emc/browse-ingredients, respectively. We used text matching and manual curation to identify active ingredients available only in the U.K., eliminating those that were already approved or generally recognized as safe by FDA (and alternate salt forms thereof) or available over the counter in the U.K. We also eliminated various non-therapeutic agents, including diagnostics, cosmetics, food additives, allergy testing antigens, and herbal and nutritional agents (including oils and vitamins). Finally, we excluded vaccines, biologics, cancer therapies, and any drug under active development by its originating company. For the remaining 133 drugs (see Supplementary File 1), we searched clinicaltrials.gov and PubMed for positive efficacy data in a new (currently off-label) orphan indication.\n\nd U.S. FDA Regulatory Procedures Manual, Ch. 9: Import Operations and Actions.\n\n\nReferences\n\nBrooke MH: A randomized trial of deflazacort and prednisone in Duchenne muscular dystrophy: efficacy and toxicity [abstract]. Neurology. 1996; 46: A476. Reference Source\n\nGriggs RC, Miller JP, Greenberg CR, et al.: Efficacy and safety of deflazacort vs prednisone and placebo for Duchenne muscular dystrophy. Neurology. 2016; 87(20): 2123–2131. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHakim A, Ross JS: High Prices for Drugs With Generic Alternatives: The Curious Case of Duexis. JAMA Intern Med. 2017; 177(3): 305–306. PubMed Abstract \| Publisher Full Text\n\nOng KT, Perdu J, De Backer J, et al.: Effect of celiprolol on prevention of cardiovascular events in vascular Ehlers-Danlos syndrome: a prospective randomised, open, blinded-endpoints trial. Lancet. 2010; 376(9751): 1476–1484. PubMed Abstract \| Publisher Full Text\n\nLarochelle M, Downing NS, Ross JS, et al.: Assessing the potential clinical impact of reciprocal drug approval legislation on access to novel therapeutics in the USA: a cohort study. BMJ Open. 2017; 7(2): e014582. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBerman A, Lee T, Pan A, et al.: Curbing unfair drug prices: A primer for states. Yale Global Health Justice Partnership, August 2017. Reference Source" }	[ { "id": "30142", "date": "24 Jan 2018", "name": "Alexander Tabarrok", "expertise": [ "Reviewer Expertise economics", "pharmaceutical regulation" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nMassive increases in the prices of some drugs have generated significant outrage and media attention. Most infamously, Turing Pharmaceutical's increase in the price of Daraprim from $13.50 to $750 made headlines around the world. In part, no doubt, because the antics of Turing CEO Martin Shkreli made him an impossible to ignore villain straight out of central casting.\nIn this paper, David and Dixit look at a seemingly similar case, Marathon Pharmaceutical's decision to price Emflaza at $89,000 a year when it is available elsewhere in the world at a price on the order of $1500 a year. David and Dixit caution that the two cases need to be distinguished because prior to Marathon, Emfalza was not for sale in the United States at any price. Marathon performed a useful service in seeking and obtaining FDA approval and there has to be a balancing of costs and benefits if we want to continue to incentivize firms to look for other pharmaceuticals from around the world and make them available in the United States.\nI agree with David and Dixit's analysis but I would go further. While David and Dixit suggest that we need \"bespoke solutions\" I worry that in trying to solve every problem we will create a mass of regulations that raise costs and harm American patients overall. We need to look beyond the headlines at the bigger picture.\nAlthough a handful of generics have seen extreme increases in prices, the prices of generics overall have been falling. The Department of Health and Human Services in 2016 concluded, for example, that big price increases were confined to small markets and that overall total spending and prices were falling.\nhttps://aspe.hhs.gov/pdf-report/understanding-recent-trends-generic-drug-prices\nMore references here:\nhttp://marginalrevolution.com/marginalrevolution/2016/02/the-good-news-on-generic-drugs.html\nThe baby in this case is much bigger and more valuable than the bathwater so even though massive increase in prices feel unjust and unfair we need to be extra careful not to throw the baby out with the bathwater. The rules governing the US generic markets are working very well. US prices for generics are low by world standards and availability is high.\nBranded pharmaceuticals in the United States are, of course, very expensive. But what needs to be stressed is that by a time a drug is approved, patents last only about 10-15 years and then prices fall rapidly with generic competition. Moreover, a large majority of prescriptions are for generics. Even more importantly, it's vital that we continue to produce new drugs to reduce morbidity and mortality. In any welfare comparison, high prices are a small price to pay for better life-saving and live-improving medicines.\nThus, I agree with the David and Dixit analysis but suggest that broadening the picture would strengthen their argument even further.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "30139", "date": "21 Feb 2018", "name": "Rena M. Conti", "expertise": [ "Reviewer Expertise Health economics", "pharmaceutical policy" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOn the topic of high drug pricing in the US, the authors present an interesting case and thoughtfully discuss it's potential implications.\nI appreciate their attempt to quantify the prevalence of future potential cases, but felt this effort could have been more systematic and involved other OECD countries.\n\nOn motivation, I think it would have been preferable article construction to start with the problem of the pricing of new drugs in the US, particularly orphans, and then located this as a \"special\" case.\n\nGenerally, the authors could have better described the tradeoffs presented in this case regarding access - entry facilitates wider access to a drug that may help American patients suffering from disease, but also creates access and affordability concerns due to the aggressive pricing practices of it's manufacturer.\n\nWithin this context, the authors insight that this case highlights the special role the FDA may play in altering pricing incentives drug manufacturers face in the US is good and particularly helpful.\nSome of the terminology used in the article to discuss the current political debate regarding high prices - notably the public pursuit of \"punishing\" manufacturers for high prices - is a bit too dismissive and could be revised. Besides from naming and shaming through the press and Congressional hearings, there has been little \"punishment\" doled out to manufacturers that pursue such aggressive pricing behavior. What punishment exists is related to pricing practices that are specifically illegal as defined by laws and statutes. The main point here should be that the manufacturer's aggressive pricing practices may hurt American patients' access to care and presents real tradeoffs to payers; what to do about this, if anything, is a real concern for patients, their families and policymakers.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "32159", "date": "22 Mar 2018", "name": "John L. LaMattina", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors discuss an important issue regarding the pricing of drugs. The issue is presented clearly and concisely, and is put in the context of other drug pricing situations where egregious prices are being charges. The authors then propose a solution to prevent such an example from occurring in the future.\n\nIt is important for this paper to be indexed as it will spark a debate about how to prevent a company like Marathon from exploiting loopholes in the US orphan drug approvals process.The Emflaza case is one where a company is charging far too high a price for an important DMD drug which has been available for decades in Europe for far less money. Furthermore, the authors show that this is not a unique situation and that it is likely to be repeated by another company. Thus, a solution is needed.\n\nThe authors propose a potential solution which should be aired and debated. Thus, I fully endorse indexing of this paper.\n\nHaving said that, I am not fully on board with the authors' solution. Marathon DID spend $50 million on clinical studies to prove the medical viability of Emflaza in DMD patients. Yes, the drug is being used off-label in Europe for DMD, but have studies been published justifying its use? If yes, how vigorous are these studies? Depending on the answers to these questions, one could argue that Marathon did a service for physicians and patients by carrying out a phase 3 program that was of sufficient rigor to garner FDA approval.\n\nThe authors' solution could work had the NIH carried out these studies and proved the value of Emplaza, thereby justifying the decision to allow Americans to import generics.\n\nAgain, my questions only serve to justify indexing of this paper to spur such debate.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-74
https://f1000research.com/articles/7-73/v1	17 Jan 18	{ "type": "Research Article", "title": "Shared genetic requirements for ATF5 translation in the vomeronasal organ and main olfactory epithelium", "authors": [ "Ryan P Dalton" ], "abstract": "Background: Both olfactory sensory neurons (OSNs) and vomeronasal sensory neurons (VSNs) require the transcription factor Atf5 for maturation and survival. In OSNs, ATF5 translation is controlled by olfactory receptor (OR) expression-mediated activation of the PERK branch of the unfolded protein response. This study evaluated whether OSNs and VSNs share genetic requirements for ATF5 translation. Methods: ATF5 immunoreactivity was assayed in whole vomeronasal organs from a series of genetic mutant animals identified in studies of OR gene choice, OR feedback, and regulation and OSN development. Results: ATF5 expression in VSNs required the histone demethylase Lsd1, which has been previously reported to be required for OR expression. ATF5 expression also required PERK-mediated phosphorylation of the translation initiation factor eIF2a. Finally, unlike previous observations in OSNs, ATF5 was found to be widespread in the mature VNO and co-expressed with mature VSN markers. Conclusions: These data suggest that the initiation of ATF5 translation in VSNs and OSNs is under similar regulation, and that persistent/prolonged ATF5 translation in VSNs may serve VSN-specific gene regulatory programs. This study firmly establishes the unfolded protein response as a major controller of sensory neuronal maturation and diversification.", "keywords": [ "olfaction", "cell biology", "atf5", "vomeronasal organ", "olfactory receptor", "vomeronasal receptor" ], "content": "Introduction\n\nMice possess two olfactory organs: the main olfactory epithelium (MOE), which houses olfactory sensory neurons (OSNs), and the vomeronasal organ (VNO), which houses vomeronasal sensory neurons (VSNs)1. The MOE is thought largely to function in the detection of odors with no prior or innate behavioral importance, though there are notable exceptions2–5. The VNO, on the other hand, detects pheromones, which drive important social and reproductive behaviors6,7.\n\nThe VNO is a bilobal, cresecent-shaped neuroepithelium. It is neurogenic, giving rise to new VSNs throughout the life of the animal1,8. Immature VSNs are located at the tissue margins, or the tips of the crescents, while mature VSNs occupy more central areas. VSNs can be initially divided into two types, based on the expression of their primary signaling G proteins. Apical VSNs express the G protein Gnai2, as well as type I vomeronasal receptors (V1Rs). Basal VSNs express the G protein Gnao and type II vomeronasal receptors (V2Rs)9,10. V1Rs are expressed monogenically and monoallelically6,11,12. The situation is markedly more complicated for V2Rs. Type II VSNs express two V2Rs in non-random combinations: one from V2R family A, B, or D; and at least one from V2R family C13,14. In addition, some basal VSNs also express at least one gene from the non-classical MHC H2-Mv gene family15,16.\n\nThe VR(s) expressed by a VSN both drive its pattern of connectivity to the accessory olfactory bulb and define its receptive field12. Therefore, the choice of receptor(s) to express is considered to be a central gene regulatory decision in VSN development. It is thought that the V1R expressed by a given type I VSN is chosen stochastically during development1. In the case of type II VSNs expressing multiple V2R genes, given that the coexpressed receptors (and H2-Mv genes for basal type II VSNs) occur in non-random combinations, it is possible that the initial V2R choice event is stochastic but acts to restrict subsequent V2R or H2-Mv choice events.\n\nThe past decade has seen the discovery of many molecular players involved in the establishment of monogenic OR expression. Monogenic OR expression begins with OR gene choice, a complex process involving condensation of OSN chromatin17, extensive modification of the OR gene chromatin environment18,19, recruitment of cis and trans enhancer elements20,21, and cooperation between a number of transcriptional activators22,23. OR choice is followed by OR feedback, which functions to preclude further OR gene choice, to promote maturation of the OSN, and to stabilize expression of the chosen OR1,24–29,30. Together, OR choice and OR feedback ensure that each mature OSN expresses exactly one OR allele, defining each OSN as a sensitive and unambiguous signaling unit.\n\nIt was recently shown that ORs drive feedback by activating the unfolded protein response (UPR), a ubiquitous signaling pathway that homeostatically maintains the ER folding environment by modifying both its folding load and its folding capacity24,31. OR expression activates the ER-resident kinase PERK, which then drives phosphorylation of the translation initiation factor eif2α, resulting in global attenuation of mRNA translation initiation and a specific increase in translation of mRNA encoding the transcription factor Atf5. ATF5 is required for OSN maturation and expression of adenylyl cyclase 3 (AC3). AC3 expression suppresses activity of a histone demethylase required for OR choice, LSD1. If OR choice fails to drive Adcy3 expression, as is the case with some OR pseudogenes as well as Atf5 and Adcy3 mutants, expression of the chosen OR is extinguished. This phenomenon, termed ‘gene switching’27, appears to add a layer of quality control for ORs, and also indicates that OR choice is initially unstable. This lack of stability is probably due to the dual demethylase activities of LSD1, which presumably allow it to de-silence an OR allele, and then to re-silence the same allele19. In this model, LSD1 downregulation by AC3 is required for stable transcription of the chosen OR. Together, these data support a model in which OR feedback acts to promotes OSN maturation, to prevent further OR choice, and to stabilize expression of the chosen OR allele.\n\nIn contrast to the growing body of knowledge on OR gene regulation, comparatively little is known for VRs. However, a number of lines of evidence support a model in which both V1Rs and V2Rs employ a feedback signal similar to that used by ORs to prevent further VR gene activation. First, VSNs choosing a V1R pseudogene target axons widely across the accessory olfactory bulb, indicating that they have subsequently selected a second V1R gene. This finding suggests that V1R protein activates VR feedback. Second, VSNs that choose an OR gene knocked into a V1R gene locus do not express additional V1Rs. This result suggests both that canonical V1R signaling is unimportant—as ORs and V1Rs signal through different second messengers—and that ORs can activate VR feedback12. Third, heterologous V2R expression activates the UPR, and both V1Rs and V2Rs, like ORs, fail to traffick from the ER when expressed heterologously. V2R trafficking appears to involve replacement of the ubiquitous chaperone Calreticulin with a VNO-specific homolog, Calreticulin 4. For V1Rs, the mechanism of ER trafficking in VSNs has yet to be established, but does not appear to involve either Calreticulin 4 or the OR transporters Rtp1/232. Finally, it was recently shown that Atf5 is required for maturation and survival of basal VSNs. This study also showed that while Atf5 mRNA expression is ubiquitous in VSNs, ATF5 protein is expressed in more limited patterns, suggesting that Atf5 mRNA is under translational control in the VNO33. In sum, these data suggest that OR and VR feedback may employ a common framework, converging on PERK-driven translation of ATF5.\n\nIn order to begin to define the mechanistic outline of VR feedback, I have assayed ATF5 protein expression in a series of mouse mutants previously employed in studies of OR feedback. I have found that in Lsd1 mutant VNOs, ATF5 protein is absent, establishing a common genetic requirement for Lsd1 in ATF5 translation in both the VNO and the MOE. Appearance of ATF5 also required both the ER-resident kinase PERK and phosphorylation of the translation initiation factor eif2α, suggesting that ER stress drives ATF5 translation in basal VSNs. Finally, in adult animals, ATF5 is widespread and found in anatomical areas corresponding to both immature and mature VSNs, suggesting that mature VSNs experience continued or spurious ER stress events. Together, these results support a model in which V1Rs and V2Rs both employ ER stress-mediated feedback, potentially with different requirements for ATF5 and subsequently with different transcriptional outcomes.\n\n\nResults\n\nLsd1 has previously been deleted from the olfactory placode by crossing animals carrying loxP-surrounded Lsd1 alleles to animals expressing Cre recominase under the control of the FoxG1 promoter. These conditional mutants lose expression of most OR genes, resulting in a failure to translate ATF5 and a failure of OSNs to reach maturity19. To test whether VSNs and OSNs share a genetic requirement for Lsd1 in the ATF5 expression, ATF5 immunoreactivity was assayed in control (FoxG1-Cre; Lsd1 fl/+) and mutant (FoxG1-Cre; Lsd1 flfl) VNO at embryonic day 18.5 (E18.5). A later analysis was not possible due to the perinatal lethality of this combination of alleles. As can be seen in Figure 1A–B, control animals exhibited robust ATF5 immunoreactivity in the VNO. As previously described, ATF5 expression was found to be widespread and heterogeneous from cell to cell. In contrast, Lsd1 mutants did not have observable ATF5 expression (Figure 1C–D). Consistent with previous findings showing that Atf5 is required for VSN maturation and survival, the VNO of the mutant animals was greatly reduced in size. Despite its decrease in size, the VNO was still readily identifiable through the use of a number of structural features, including the surrounding bone and mesenchyme structure, bilateral symmetry, position relative to the MOE, and the presence of a lumen of stereotypical shape, adjacent to an epithelium with a single layer of apical sustentacular cells. Together, these data indicate that in the VNO Lsd1 is required for ATF5 expression, and by extension for VSN maturation. On the basis of these data, I hypothesize that VR expression is under Lsd1 control, and that VR expression drives ATF5 translation. This hypothesis will be addressed in further detail in the discussion section.\n\n(A–B) Representative coronal section of embryonic day 18.5 (E18.5) VNO from a FoxG1-Cre; Lsd1fl/+ animal. (C–D) Representative coronal section from a FoxG1-Cre; Lsd1 fl/fl VNO, also stained for ATF5 and DAPI. For all images, ATF5 immunoreactivity is shown in red and DAPI nuclear counterstain in blue.\n\nI next asked whether ATF5 translation in the VNO is under the same regulatory control as in the MOE. The Atf5 mRNA contains an inhibitory upstream open reading frame (iuORF) that under basal conditions suppresses its translation. However, upon phosphorylation of the translation initiation factor eIF2α at Serine-51, ribosomes bypass this iuORF to translate the Atf5 mRNA coding sequence24,34–37. OR expression in the MOE promotes this phosphorylation event and ATF5 translation by activating the ER-resident kinase PERK. OR-driven ATF5 translation can be blocked either through PERK deletion or through mutation of the serine phosphorylation site on eIF2α to alanine. I therefore asked whether ATF5 was lost in the VNO of PERK mutants and eIF2α phosphor-mutants. While P0 Perk+/- VNO exhibited robust ATF5 immunoreactivity (Figure 2A–B), ATF5 was completely absent in littermate Perk-/- animals (Figure 2C–D). Similarly, ATF5 was completely absent in eIF2αS51A/S51A animals, in which PERK is still present but cannot exert translational control through eif2α phosphorylation (Figure 2E–F). These data indicate that, as has been observed in the MOE and elsewhere, Atf5 mRNA in the VNO is under translational regulation via PERK-dependent phosphorylation of eIF2α.\n\n(A–B) Coronal section of postnatal day 0 (P0) VNO from Perk+/- animal. (C–D) Coronal section of P0 VNO from a Perk-/- littermate. (E–F) Coronal section from a PO eIF2α S51A/S51A animal. For all images, ATF5 immunoreactivity is shown in red and DAPI nuclear counterstain in blue.\n\nIn the MOE, ATF5 expression is restricted to immature OSNs24. This expression pattern is intriguing, as both Atf5 and OR mRNA continue to be expressed in mature OSNs. It has been proposed that this context-dependence for ATF5 translation is due to increased expression of OR transporters such as RTP1/2 in mature OSNs, which could compete ORs away from PERK or simply relieve the ER burden imposed by ORs. In the VNO, a previous report demonstrated that at P0, Atf5 mRNA expression is essentially homogenous across the neuronal area, but that ATF5 protein is more heterogeneous. However, this report did not assay ATF5 expression in adult animals. While in young animals the VNO and MOE are dominated by immature neurons, in older animals the immature and mature neuronal compartments separate and resolve. In the adult VNO, a number of reports, using a variety of markers, have shown that immature VSNs are restricted to the VNO margins (i.e. the ‘tips’ of the VNO crescents)38,39. Surprisingly, ATF5 was not restricted to the tissue margins in the adult VNO. Instead, it was widespread, heterogeneous, and found in areas corresponding to both immature and mature VSNs (Figure 3A–B). Furthermore, co-staining sections from this animal with antibody against olfactory marker protein (OMP) to label mature VSNs revealed that some ATF5-labeled cells co-express OMP. While most OMP+ cells were either ATF5-negative, or displayed barely-detectable ATF5, other OMP+ cells displayed saturating levels of ATF5. These observations suggest that, unlike in the MOE, in VSNs ATF5 continues to be expressed after maturation. Given the nature of ATF5 translational control and the consequences of persistent UPR activation, this raises a number of interesting questions regarding PERK activation dynamics and the transcriptional output of ATF5 in VSNs.\n\n(A–B) Coronal section of a postnatal day 35 VNO. ATF5 immunoreactivity is in red and DAPI in blue. (C–F) A close-up section from the same VNO as shown in A–B. Olfactory marker protein (OMP) immunoreactivity is shown in green, ATF5 in red, and DAPI in blue.\n\n\nDiscussion\n\nReceptor-driven feedback programs endow developing olfactory neurons with a means by which to establish distinct, unambiguous cell fates. These programs are therefore essential in the construction of the basic architecture of the olfactory system. Furthermore, because these feedback programs allow the appearance of a single protein to establish cell fate, they also act as an engine in neuronal diversification, forging a direct relationship between the number of chemoreceptor genes and the number of chemosensory cell fates.\n\nMy previous work uncovered that in OSNs, OR feedback is executed by co-option of the PERK branch of the unfolded protein response24. An obvious follow-up question to this work was whether this feedback mechanism was employed in other tissues in which sensory cells express single or small numbers of sensory receptors. In the present work, I demonstrate that expression of ATF5, which is required for OR feedback and for the maturation and survival of basal VSNs33, has the same genetic requirements in the VNO and the MOE. This work therefore strongly suggests that ORs and VRs have a shared mechanism of feedback, converging on activation of the PERK branch of the UPR. This work was undertaken at the tissue level, and therefore more detailed analyses will likely be required in order to determine the specific requirements of VSN subtypes. Below I discuss some of the caveats of this work, as well as interesting questions for future work.\n\nFirst, I hypothesized above that VR expression is under Lsd1 control and is required for ATF5 expression. Several pieces of data prompted this hypothesis. Among them are the shared elements in VR and OR feedback discussed in the introduction such as their activation of the UPR in cell lines, as well as the requirement of Lsd1 for ATF5 expression in the VNO demonstrated herein. However, it has yet to be directly demonstrated whether and how Lsd1 influences VR expression, or whether VR expression in VSNs drives ATF5 translation. A number of experimental considerations make these analyses difficult. Among them are the prenatal lethality of Lsd1 mutants and the requirement of Atf5 for VSN survival19,33, which together result in exceedingly small amounts of tissue for analysis of VR expression or of the epigenetic landscape of the VR gene family. Additional genetic models would likely be useful in determining the role of Lsd1 in VR choice, and a combination of biochemical and genetic approaches would be powerful in the determination of the mechanisms by which chemoreceptors influence PERK activity.\n\nSecond, it has been shown that a key element of OR feedback is AC3-driven downregulation of LSD119. In the MOE, LSD1 downregulation both prevents further OR choice and acts to stabilize expression of the chosen OR. LSD1 downregulation therefore must be exquisitely timed. No analogous situation has yet been demonstrated for VSNs. It is worth noting that the requirements for VSNs are likely different than for OSNs. In particular, VSNs choosing VR pseudogenes continue to express them while also selecting another VR from a different VR gene cluster40. This finding indicates that VR choice may involve the permanent engagement of a single or limiting element in cis to a given VR cluster. VR feedback may therefore act to prevent further choice, but not to stabilize VR expression. Thus, if VSNs employ a mechanism similar to that of AC3 in OSNs, it may only act to terminate further VR choice, but not to stabilize VR choice. The mechanistic basis of this difference is a fascinating area for future study.\n\nThird, the convergence on ATF5 in OSNs and VSNs prompts a number of questions on the role and transcriptional output of ATF5. For example, how could ATF5 control OR feedback in OSNs and VR feedback in VSNs? It seems likely, given that ATF5 is a bZip-family transcription factor, that ATF5 has different binding partners in different tissues. This model would allow for co-factors to tune the transcriptional specificity of ATF5, but would prevent their engagement until ATF5 has been translated. For example, in OSNs this may allow ORs to promote expression of RTP1/2 such that they can subsequently be targeted to the plasma membrane. In contrast, given that basal VSNs express non-random combinations of receptors and that the expression of these receptors is sequential, expression of one VR may drive ATF5 expression to aid in selection of a second VR (or an H2-Mv). The identity of these potential binding partners is a fascinating outstanding question and is likely to greatly aid in our understanding of chemoreceptor feedback programs.\n\nFourth, as demonstrated herein, ATF5 continues to be expressed in mature VSNs, unlike findings in OSNs. In addition, cell-to-cell levels of ATF5 appeared to be extremely variable, with signal nearly undetectable in most cells, but reaching saturation levels in other cells. This is a fascinating observation, as it would indicate that mature VSNs continue to experience ER stress events. Atf5 is ubiquitous in VSNs33 and the UPR-driven mRNA translation program is rapidly induced but brief. I therefore hypothesize that the ATF5 expression patterns I observed reflect transient ER stress events experienced by many or all VSNs. However, it is impossible to rule out an alternate scenario in which some cells (or even VSN sub-types) experience continuing ER stress while others do not experience ER stress at all. An additional implication of the prolonged ATF5 expression pattern in VSNs could be that VRs and ORs have different mechanisms of PERK activation, for example direct versus indirect. A number of studies support an indirect model of PERK activation by ORs, in which ORs activate PERK only in the absence of RTP1/2, but this question is unaddressed for VRs. In addition, it is intriguing that ATF5 could be continuously expressed in mature VSNs, as it would beg the question of how VSNs can differentiate between bona fide ER stress and this developmental signal. Whether ATF5 has direct anti-apoptotic functions in VSNs as has been observed in other cell types has yet to be determined.\n\nFinally, these findings firmly establish that a pathway canonically thought to be involved in the detection and resolution of cellular stress responses is fundamental in the designation of cellular identity and in cell maturation. This not only begs a reassessment of the role of PERK signaling, but also suggests specific additional studies. Given that activation of PERK provides such a powerful means by which to coordinate receptor appearance to the cellular gene expression program, and given that a multitude of cell types are defined by their expression of one or a handful of receptors, it would be surprising if PERK were not involved in other receptor-driven feedback programs. Excellent candidates include somatosensory neurons expressing Mas-related GPR family members, taste receptor cells, and photoreceptor cells. Specific chaperone or transporter requirements for these different receptors would provide a simple and generalizable mode for receptors to activate PERK in order to drive global gene expression programs, whose outputs can then be tuned by the use of tissue or cell type-specific co-factors.\n\n\nMethods\n\nAll mice were housed in standard conditions with a 12-hour light/dark cycle and access to food and water in a UCSF barrier facility. All mouse experiments as well as euthanasia were approved by and were in accordance with University of California, San Francisco Institutional Animal Care and Use Committee (IACUC) protocol as described previously18. Animals used in this study were under protocols held by the Lomvardas laboratory. Details on standard procedures including euthanasia can be found at the UCSF IACUC website (http://iacuc.ucsf.edu/Policies/awStandardProcedures.asp). Because all animals described in this study were only used for tissue collection, the relevant UCSF IACUC sections are those that deal with proper euthanasia. For all animals used in this study, animals were single or pair-bred (for animals harvested during pregnancy) or were group housed (for animals harvested as adults). For prenatal experiments, pregnancies were timed such that pregnant females and pups could be harvested at the time points listed in the study. The age at which tissue was collected for each experiment is indicated in the figure legends. For all tissue collected, animals were euthanized at the indicated time point using a CO2 flow rate designed to minimize suffering and per IACUC regulations. Following CO2 exposure, animals were secondarily euthanized either by cervical dislocation for adults or decapitation for prenatal and perinatal pups, and then immediately dissected. Main olfactory epithelia or vomeronasal organs were dissected using clean, sterile, and recently sharpened tools.\n\nAll strains were maintained on a mixed (Black 6 / FVB) genetic background. The following mouse lines used in this study have been previously described: FoxG1-Cre; Lsd1 flox19, Perk and Eif2αS51A/S51A24. No sex-based differences in expression of the markers tested herein were identified in preliminary studies and the studies herein contained animals of both sexes. All efforts were made to minimize the number of animals used in performing this study.\n\nImmunofluorescence (IF) was performed as previously described17,19,24. All animals were dissected immediately following euthanasia by CO2 exposure. Briefly, tissue was directly dissected into optimal cutting temperature compound (OCT, Tissue-Tek #4583). 14µm sections were air-dried on glass slides (VWR #48311-6703) for 10 minutes, fixed in 4% paraformaldehyde (PFA, Sigma #158127) in phosphate buffer solution (PBS) for 10 minutes, washed 3x5 minutes in PBS + .1% Triton-X (PBST), blocked for 1 hour in 4% donkey serum in PBST, then incubated with primary antibodies diluted in PBST and under coverslips (VWR # 470019-008) overnight at 4C. The following day, slides were washed 3x15 minutes in PBST and then incubated with secondary antibodies and DAPI in PBST at concentrations of 1:1000 under cover slides. Slides were then washed 3x15 minutes in PBST and mounted with vectashield (Vector Laboratories # H-1000) for imaging. Imaging was performed on Leica 700-series laser scanning confocal microscopes. The following antibodies were used: goat anti-Atf5 (Santa Cruz Biotechnology, SC-46934, dilution 1:250), rabbit anti-OMP (Abcam ab93127, dilution 1:250). For each panel, at least one mouse per genotype was sectioned. Differences were not noted between males and females. Mice were genotyped in-house using genotyping protocols suggested by the original generators of the mouse line. Genotyping protocols included positive and negative controls reactions. Full MOE or VNO were sectioned. To minimize variability between slides, control and experimental genotypes were sectioned onto the same slide. Slides with strong antibody signal and low background were selected for analysis. For each section, microscope settings were optimized for signal:noise. All image analysis was done in Fiji (version 2.0.0-rc-39/1.50b, build 8dcf1e65a6) and consisted only of changing brightness and contrast for each channel.\n\nAll efforts were made to ameliorate the suffering of animals used in this study. Animals received regular care from the author and from the animal facility personnel, as well as monitoring for health and injury. Unhealthy or injured animals were either treated or, in severe cases, humanely euthanized. In accordance with UCSF IACUC guidelines (see above), all animals were euthanized by exposure to CO2 at a rate deemed to minimize stress and suffering. Animals were not bred unnecessarily, and when possible multiple types of tissue were dissected from each animal and saved for potential future use.\n\nDataset 1: Raw images of immunofluorescence presented in figures 10.5256/f1000research.13659.d19056141", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by a Genentech Predoctoral Fellowship\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nDalton RP, Lomvardas S: Chemosensory receptor specificity and regulation. Annu Rev Neurosci. 2015; 38: 331–349. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuck L, Axel R: A novel multigene family may encode odorant receptors: a molecular basis for odor recognition. Cell. 1991; 65(1): 175–187. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnomoto T, Ohmoto M, Iwata T, et al.: Bcl11b/Ctip2 controls the differentiation of vomeronasal sensory neurons in mice. J Neurosci. 2011; 31(28): 10159–10173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoppolo D, Vollery S, Kan CD, et al.: Gene cluster lock after pheromone receptor gene choice. EMBO J. 2007; 26(14): 3423–3430. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDalton RP: Dataset 1 in: Shared genetic requirements for ATF5 translation in the vomeronasal organ and main olfactory epithelium. F1000Research. 2018. Data Source" }	[ { "id": "31794", "date": "21 Mar 2018", "name": "Paolo E. Forni", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe research presented by Dalton aims to investigate mechanisms that control the expression of vomeronasal receptors in mice. The paper is very clear, well written and analyzes a relevant question in the field. Moreover, the author nicely discusses how his findings could be relevant for the study of different types of neurons. Selective expression of olfactory neuron receptors (OR), in the main olfactory epithelium, relies on a complex mechanism that include epigenetic DNA modification and interaction of different transcriptional regulators. Olfactory receptor expression activates the ER-resident kinase PERK, which controls phosphorylation of the translation initiation factor eif2α, and increase in expression of the transcription factor ATF5. ATF5 expression controls Adenylate cyclase 3 expression. In the main olfactory epithelium, it has been proposed that the OR selective choice depends on removal of silencing chromatin modifications by the demethylase Lsd1 and that the adenylate cyclase 3 expression blocks LSD1 activity. Previous work (Nakano et al 20151) has shown that ATF5 is important for maturation and survival of basal vomeronasal sensory neurons. Based on these premises Dalton proposes that vomeronasal receptors share a similar expression feedback mechanism, to the one described in the MOE, based on ER-resident kinase PERK activation, EIF2α phosphorylation and ATF5 translation. To understand if a similar regulatory network, to the one described in olfactory neurons, exist in the VNO Dalton analyzed ATF5 expression in Foxg1Cre;Lsd1flox/flox , Foxg1Cre;PERKflox/flox conditional mutants and in mice carrying a mutated form of eIF2α (eIF2α S51A/S51A) that prevents its phosphorylation and therefore AFT5 expression.\n\nMajor points:\n-In line with the hypothesis ATF5 expression is lost in the vomeronasal epithelium of Lsd1, PERK and eIF2α S51A/S51A mutants suggesting that ATF5 is under control of PERK, Lsd1 and EIF2a. However, while the PERK mutants appear to have a relatively normal vomeronasal epithelium in Lsd1 and eIF2α mutants the VNO appears to be almost vestigial suggesting that these genetic manipulations affect much more than ATF5 expression alone. The phenotypes of Lsd1 and eIF2α mutants do not overlap with the one described in the ATF5 conventional KO (see Nakano et al. 20151). In fact in the ATF5 KOs a reduction has been described in basal vomeronasal neurons only, in the Lsd1flox/flox and eIF2α S51A/S51A mutants all the vomeronasal neurons seem to be affected. Are there vomeronasal genes that are not affected after Lsd1 and eIF2α S51A/S51A mutations? Is proliferation altered? Are the neurons dying? All mutants should be presented including at least GAP43 and OMP, Ki67 and activated Caspase-3 immunostaining. Though the lack of ATF5 expression is in line with the main hypothesis more internal controls are needed to support the conclusion.\n\n- Dalton nicely shows that ATF5 is expressed in mature vomeronasal neurons. Is ATF expression still controlled by Lsd1, PERK and eIF2α in mature (OMP+) vomeronasal sensory neurons? Characterizing ATF5 expression after OMPCre drive deletion of Lsd1 and PERK would strengthen the quality of the paper and its relevance.\n-The number of analyzed animals is not clearly indicated.\n\n- Heterozygous Foxg1-cre mice can have developmental phenotypes (Eagleson et al. 20072) ATF5 expression should be also analyzed in FoxG1Cre+/- controls.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "32156", "date": "23 Mar 2018", "name": "Gustav F. Jirikowski", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a morphological study on histochemical similarities between vomeronasal and MOE sensory neurons in mouse during pre- and postnatal development with regard to the distribution of transcription factor Atf5. The expression of ATF5 depends on the histone demethylase Lsd1. ATF5 staining of samples from Lsd1-mutants revealed negative results. The expression of ATF5 also seems to require PERK. There was no ATF5 staining seen in Perk -/- mutants. The observations are original, interesting to a wide audience and deserve publication. However a few issues need to be addressed prior to final acceptance of this paper: Photomicrographs need to be presented at higher magnifications and in higher quality. The overall morphology of the mouse VNO is most likely known to the majority of readers. So one low power image may be enough to give an overview. All other pictures should be at higher magnifications in order to reveal the details described in the results section. Micrographs showing ATF5 staining alone can be omitted since sections counterstained with DAPI provide the same information. It seems that ATF5 is confined to the sensory epithelium only in newborn mice. In both E 18,5 and PNE 35 animals ATF5 seems to occur also in cells of the non sensory epithelium. This observation needs to be mentioned in results and discussion. This is mere histochemical study. Neither physiological nor molecular data are presented. Therefore these aspects need to be discussed more cautiously. Minor points:\np.2, line 1: There are more than 2 olfactory organs in rodents: In addition to the MOE and the VNO there are Grüneberg ganglion and, Septal organ. p.2, line 3-4: The introduction also need to mention the non-sensory epithelium of the VNO p.2, line 12: A reference is necessary here. p.2, line 36: The definition of the abbreviation OR is missing. p.7, methods: How many mice were examined? There are still a few typos throughout the text.\nLettering of figures (small letters) should match lettering in captions (capital letters).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "32153", "date": "27 Mar 2018", "name": "Pelin Cayirlioglu Volkan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study from Dalton aims to understand whether the same mechanisms that control olfactory receptor (OR) gene choice also govern the selection of vomeronasal receptors (VRs). The paper presents several immunohistological assays demonstrating that the expression of ATF5 is regulated by similar genes to those observed in the olfactory epithelium. Finally, the author shows that the expression pattern of ATF5 in the vomeronasal organ is distinct from the observed pattern in the olfactory epithelium.\nOverall, this work is well presented and clearly described. A few points deserve revision. Major points: The author acknowledges that there is significant cell death caused by mutation of Lsd1. An additional control demonstrating that the expression of a housekeeping gene would be useful to demonstrate that gene expression is not globally altered in the surviving cells.\nMinor points: The statement of a hypothesis in the Results section should be deleted. This hypothesis is adequately addressed in the Discussion section.\nLines delineating regions that contain immature vs mature VSNs could be drawn in Figure 3 to aid readers who are less familiar with the structure of the VNO.\nThe second to last paragraph of the introduction lacks several references. The authors should review this paragraph and add citations.\nThe sentence beginning \"A number of studies support...\" In the second to last paragraph of the discussion lacks a citation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-73
https://f1000research.com/articles/7-72/v1	17 Jan 18	{ "type": "Opinion Article", "title": "Rival perspectives in health technology assessment and other economic evaluations for investing in global and national health. Who decides? Who pays?", "authors": [ "Anthony Culyer", "Kalipso Chalkidou", "Yot Teerawattananon", "Benjarin Santatiwongchai", "Kalipso Chalkidou", "Yot Teerawattananon", "Benjarin Santatiwongchai" ], "abstract": "There seems to be a general agreement amongst practitioners of economic evaluations, including Health Technology Assessment, that the explicit statement of a perspective is a necessary element in designing and reporting research. Moreover, there seems also to be a general presumption that the ideal perspective is “societal”. In this paper we endorse the first principle but dissent from the second. A review of recommended perspectives is presented. The societal perspective is frequently not the one recommended. The societal perspective is shown to be less comprehensive than is commonly supposed, is inappropriate in many contexts and, in any case, is in general not a perspective to be determined independently of the context of a decision problem. Moreover, the selection of a perspective, societal or otherwise, is not the prerogative of analysts.", "keywords": [ "HTA", "CEA", "perspective", "stakeholder", "social value judgments", "reference case", "patients’ preferences." ], "content": "Introduction\n\nIt seems a desirable thing to set international quality standards for conducting health technology assessments and other economic evaluations (EEs) of the impact and desirability of investments that promote health1. Such standards are, however, not easy to define, let alone to get agreement on2. What is easy is to fall into a trap of small-town thinking, by setting standards whose applicability is arbitrarily and quite needlessly restricted to: (a) particular cultural and political contexts, (b) within any such context, a set of choices bounded by further arbitrary value judgments, (c) a presumption that EE methods are designed for use only in the public sector – and, moreover, only when the object is “the public interest”. Parochialism has two unattractive features. First, it denies the possible usefulness of EEs outside the public sector and to non-governmental agencies, like trade unions, for-profit companies and non-profit organisations like charities. Second, it imports value judgments to the effect that (a) EE can be conducted in a context-free way without attention being given to cultural and political values that may be particular to the application, which may not be readily transferable from jurisdiction to jurisdiction and (b) that the use of patients’ (or the public’s) preferences, in constructing outcome measure such as the quality-adjusted life year (QALY) or disability-adjusted life year (DALY) via estimated or expressed consumer values, rather than using, say, judgments made by parliamentary committees, expert groups or citizens’ juries, provides an appropriate and relevant index of value. We seek to avoid such restraints by proposing that questions of perspective be determined in context, where the job of EE analysts is to help decision makers to articulate a perspective that is appropriate to the circumstances rather than to prescribe a perspective independently of its context of use (being “context free”).\n\nFirst, we review the recent history of “perspective”.\n\n\nWhat is perspective and why does it matter?\n\nGuides to best practice in cost-effectiveness analysis (CEA) can be traced back at least as far as 1980, with the publication, at the initiative of Senate Committee on Labor and Human Resources and commissioned by the then Office of Technology Assessment (OTA), of The Implications of Cost-Effectiveness Analysis of Medical Technology3. Other significant landmarks appeared in 1994 by the Canadian Coordinating Office for Health Technology Assessment4, in 1996 by Gold et al. (the “Washington Panel”)5, in 1997 by Drummond et al.6, in 2003 by the World Health Organization7, in 2004 by the National Institute for Clinical Excellence (NICE)8 and in 2016 by the second Panel9 and the International Decision Support Initiative (IDSI)10.\n\nThe Washington Panel coined the term “Reference Case” to describe the characteristics of a high quality economic evaluation, thereby establishing both a lasting piece of terminology and launching a stream of literature (see original publication here: https://www.ncbi.nlm.nih.gov/pubmed/8861994 and over 170 references in PubMed: https://www.ncbi.nlm.nih.gov/pubmed?LinkName=pubmed_pubmed_citedin&from_uid=8861994). The Panel’s work absorbed much of that by Drummond et al6. One of the characteristics of a high-quality EE was the explicit stating of a study’s “perspective”, described by the panel as “the study’s point of view”, which “determines which health outcomes and costs are relevant and plays a part as well in how they should be valued”. We wholly agree with this principle and it seems to have formed a part of all subsequent reference cases, including one developed by the Drummond et al. team and us, through the Gates-sponsored iDSI Reference Case (http://www.idsihealth.org/resource-items/idsi-reference-case-for-economic-evaluation/). The case for it is made not merely on grounds of transparency, which matters because it enables other researchers to check that the scope and measures of costs and benefits were indeed fit for purpose, but also on grounds of accountability: what to include and how to measure it are essential political and social value judgments that ought to be made by duly accountable decision makers (not EE analysts!). A good study will be, therefore, explicit about its perspective (or perspectives, if more than one is to be explored).\n\nThe Washington Panel recognised that a perspective must reflect the purpose of a study. It is not always appreciated that the perspective will usually be context-dependent, with the context determining the appropriate perspective. For example, a study might appraise the costs and benefits of a workplace intervention to reduce back strain on workers through redesigned work stations from the points of view of managers on the one hand and trade unions on the other, with a view to anticipating opposition and identifying compensatory and regulatory measures for successful implementation. In such a case, there are three perspectives to be considered – those of management, workers and government. The perspectives to be used are defined by the purpose of the study which in turn is specified by its sponsor: in this example, management, workers or the government (or a consortium of all three).\n\n\nWhen it comes to perspectives, is broader better?\n\nThe First Washington Panel’s Reference Case recommended that a societal perspective be adopted, offering decision makers as wide as possible an understanding of the costs and consequences of alternative actions both in the health care sector and beyond it into the private health care sector and non-health sectors, public or private. Its reasoning is plain: only if the societal approach is adopted can decision makers be confronted with a full information set of the costs and consequences of alternative actions. Anything less comprehensive will necessarily be subject to omitted variable bias, probably of unknown sign and size, causing either over- or under-investment in both old and new technologies. It is hard to raise an objection of principle to this. Pragmatically, however, to seek inclusion of every consequence, no matter what the costs are of tracking it down and quantifying it, seems completely over the top: the limits of inclusion ought more rationally to be determined by a sensitivity analysis of whether inclusion or exclusion makes a difference of significant concern to any relevant outcome. At any rate, reasonably full information might be conceded as a desirable thing when the objective is to produce a decision that is in the public interest. What constitutes “reasonable” in any given context would necessarily be a matter for local judgment (decision makers and advisers). If the objective were less comprehensive than societal, however, to collect full information would be to collect much that was irrelevant to the context of that decision question. The argument that “more information is always better” is therefore not an argument for adopting the societal perspective; it is an argument for EEs that can reflect many perspectives, including the “societal” one, but only up to the point at the value of further information is judged to be less than the cost of collecting and incorporating it.\n\nWhat “societal” means is, even in its most comprehensive version stated in the literature, commonly rather more restricted in terms of the nature of the consequences considered than may be thought. Compassionate externalities11–13 are usually not included. Stresses and welfare losses resulting from changes in delivery pathways, provider identity, loss of employment (temporary or permanent), limited involvement in or being informed about changes, confusion and misunderstanding of the purposes of changes – such effects characteristically accompany both investment decisions and changes in the ways in which decisions are made but none, however, feature in the scope of “societal” as commonly stated. “Societal” is thereby significantly short of being as comprehensive as may appear.\n\nIn any event, in practice, not all have followed the Panel’s recommendation. Table 1 provides an illustrative variety of approaches. As would be expected, perspectives offered by an academic do not necessarily reflect the expressed views of public agencies. Developed countries like England, Australia and Canada rely on EE to inform allocation decisions and tend to be narrower in their scope of analysis whereas global Reference Cases tend to adopt a wider perspective. Some low-income and middle-income countries (LMICs), where out-of-pocket costs are significant select a societal perspective though very few have a track record of using EE systematically.\n\nThe limits of feasibility are much more tightly drawn in LMICs than in rich countries, where a closer approximation to “full information” may be both possible and desirable. In LMICs, what is possible is much more limited due to poor or absent data, lack of technical skills to conduct and use EEs, and lack of political understanding, leadership and support14,15. Table 2 shows that a healthcare payer’s perspective is the most popular recommendation in the national guidelines of high-income countries where EE is commonly used to inform reimbursement of health services. Only a third of the guidelines from both high-income countries and LMICs recommend a societal perspective.\n\nSource: GEAR’s guideline comparison http://www.gear4health.com/gear/health-economic-evaluation-guidelines\n\nIt would also be rash to suppose that what is regarded as the public interest is always and everywhere the same. Just such a belief underlies the World Health Organization (WHO) advocacy of DALYs as a kind of universal outcome metric with weights attached to dimensions of disability that are invariant with respect to culture and context. This is advanced as an advantage of DALYs over QALYs, since QALYs typically use customized country weights. A similar insensitivity can be seen in the WHO’s abandoned attempts to recommend thresholds16. A threshold is an aggregate expression of collective willingness to pay for treatments in national health insurance. It represents a marginal preference for public expenditure on health care relative to the many other objects of public policy – poverty reduction, education and training, housing, law and order, civil and national defence, and so on. There can be no presumption that one size fits all, regardless of political stability, stage of economic development, culture, religion, or history. We should not encourage a similar insensitivity to enter an authoritative methodological guide to best practice in EE.\n\n\nSocietal perspective: Whose values and whose judgments count?\n\nA problem with the use of a societal perspective is that it raises the possibility of reintroducing into health policy decisions many of the very biases that most health care systems have been designed to avoid. A critically important one concerns the implications of a perspective for the role of valuation and willingness to pay for health gains. It is part of the conventional wisdom of health economics that the health care market is characterised by more market failures – and serious ones at that – than almost any other market. Many of these impinge on the reasonableness of using individual willingness to pay as a priority-setting criterion and, more generally, of using the Pareto criterion (or the potential Pareto criterion) to identify socially beneficial changes17. These are standard aspects of the textbook treatments of heath economics: we merely list those that are of chief concern (Table 3). It is not satisfactory, as in Pauly (1995)18 and others who advocate the use of consumer preferences as a basis for public priority setting, to glide over these issues as if they were empirically or politically unimportant. The problems of reliance on consumer preferences for healthcare relate not only to measures of individual (as distinct from collective) willingness to pay but to all expressions of patient preferences. They raise the difficult question of the right balance to be struck between individual expressions of value and collective ones.\n\nThis is not the place to argue the cases for or against individual willingness to pay as a basis for making health investments or of locating the patient as a decision maker in a healthcare system. Our purpose is only to argue that a societal perspective need not necessarily require its source of value, whether or not monetised, to be individual patients. The welfare of patients remains a central concern but avoiding the distortions of the real world is necessary if patients are to be responsibly protected.\n\nWhat, therefore, is the appropriate source for social valuations of the kind used in EE? And whose willingness to pay for the treatments offered in public insurance benefit packages of the sort aspired to, especially in LMICs, should be embodied in policy? We are not concerned here with individual choices of clinical providers and treatments, though the bulleted issues below do have implications for information provision, clinical audit and health care quality assurance. The questions for EE concern instead the value content of a set of key collective judgments that usually have to be made. The answer to all of the following will be shaped by the perspective:\n\nThe choice of outcome measure (life-years? Lives? DALYs? QALYs? Market value of human capital generated, or commercial gain?).\n\nThe construct validity of the possible measures, given the values and culture of country in question (which may themselves be heterogeneous across regions/provinces). (“Does this measure correspond sufficiently to what we collectively understand to be the underlying concept (health)?”)\n\nThe impact of the country’s budget for health care on the affordability of the various potential benefit packages (or extensions to them). (“What would a modest addition to or subtraction from the current level of public expenditure on health and healthcare imply for additional possible interventions, or for those most likely to be removed from the benefits package?”)\n\nThe relative value in real terms (opportunity cost) and at the margin of the various interventions. (“Do we need a money measure or is it sufficient to compare the impact of each on health?”)\n\nThe relative value in real terms (opportunity cost) and at the margin of extensions of entitlement. (“How do we trade-off the benefits of extending the benefits package to more people or specific groups (dwellers in remote places, workers, civil servants, the military...) against increasing the scope and size of the package for existing members of the scheme?”)\n\nThe relative value in real terms (opportunity cost) and at the margin of increased financial protection from the hazard of catastrophic (or significantly large) personal healthcare expenditure. (“Which population groups are most likely to benefit and what is a good measure of that benefit?”)\n\nThe monetary value of an increment in outcome required if an intervention is to be added to the benefit bundle or to displace existing benefits. (“What price adjustments must the manufacturer offer for this intervention to become cost-effective?”)\n\nThe collective answers to these questions require no knowledge of individual willingnesses to pay for health care. Instead they require decisions taken at the societal level by decision makers accountable through some locally legitimate political process, advised where appropriate by expert opinion and supported by the best available evidence. Individual preferences/values might be considered relevant in selecting outcome measures, their dimensions (physical, mental functioning, etc.), the weights attached to various dimensions that enable them to be added up to create an index of outcome, and the weights (commonly unity) to attach to individuals when individual levels of health or disability are aggregated to community level. Decision makers need to exercise judgment regarding the extent to which individual members of a community are to be involved in these judgments. The societal judgments that are required are themselves choices that should be context-dependent and essentially locally accountable.\n\n“Societal” does not necessarily imply “everyone”. Decision makers may have their own views about whom to consult and when. They will have their own views as to what exposures to risk and distributions of cost and benefit are fair. They may have general across-the-board financial rules for public sector investments that stipulate discount rates and time horizons. They will address at a higher level what can be afforded for health care in the light of pressing needs for education, housing, poverty relief, law and order, etc. They will decide whether their constitutional preference is to narrow the idea of health to whatever impact health care provision has on it. They will decide whether to consider non-health consequences of health care investments.\n\n\nWho are the stakeholders? A tentative list\n\nStakeholders all have perspectives, some of which may be shared, but most of which will need to be accommodated in some way in the methods of EE and the processes through which it is conducted. The extent to which individual ideas about perspective can be effectively suppressed in the interest of having a shared and publicly stated perspective has not yet, so far as we are aware, been explored empirically. EE analysts can give guidance. For example, a suggested list of possible stakeholders whose roles are for decision makers to consider (which has to be context-sensitive) might be as follows:\n\nRepresentative of the general (or relevant regional) public\n\nPatients with personal or vicarious experience of a relevant condition\n\nFamily informal carers\n\nRelevant clinical specialists\n\nClinical generalists\n\nProfessional leaders (early adopters)\n\nPoliticians (including the opposition parties)\n\nResearchers (all relevant disciplines)\n\nProduct manufacturers\n\nHealth service providers\n\nHealth service managers\n\nAid givers and donor organisations\n\nRelevant NGOs and other sponsors\n\nInsurers (public and private)\n\nEducators\n\nMedia communications specialists and journalists\n\nParliamentarians\n\nThe Judiciary\n\nOther interested parties\n\nSelection of stakeholders and possible roles they might play in determining the perspective are matters on which EE analysts may advise, for example, by listing options, sharing experience elsewhere, indicating likely consequences, and building models.\n\n\nOn the role of the analyst (and how we differ from others)\n\nOur recommendation is, then, that analysts should approach the issue of perspective at two levels of specificity: the universal or general (context-free) on the one hand, and the context-dependent on the other. A fundamental universal context-free principle is that the perspective always be stated, be consistently adhered to throughout an EE study and appear in reports of the study. Another is that the reasons for the perspective should be given, any departures from previous or common practice elsewhere noted and explained.\n\nThese two principles will also shape much of the process in conducting an EE; for example, identifying partners and stakeholders, the extent of consultation, the need for new context-specific research, time-lines and reporting accountability. Their roles will depend upon the context-specific perspective actually adopted. For example, most perspectives will doubtless need to include physicians and patients in consultation meetings, but the scope of the issues discussed will differ according to the specific perspectives: in some studies they might advise on the probable accuracy of the outcome measure and the likely cost consequences of adopting a new technology in the social care sector, in others, they may be concerned only with the outcome measure. The national payer may specify that no additional resources will be available, which will encourage a relatively narrow perspective. Alternatively, the national payer may want an EE performed in order to inform a review of public expenditure across all sectors, in which case a broader public-sector perspective is implied. A health advocacy group, by contrast, may wish a societal perspective to be adopted to establish a case for increasing the health budget. A public payer committed to health maximisation is likely to adopt a perspective that focuses narrowly on health as an outcome while being much broader in its consideration of cost-effective means of promoting health by embracing nutrition, public education, housing and other determinants not normally within the direct control of the Ministry of Health. In each case the perspective is determined by the interests of the client for the analysis. A donor committed to universal health care might adopt any of a number of positions regarding perspective, depending both on its fundamental strategic purposes (perhaps with a single disease perspective) and on the specific context of application (perhaps having specific regard to local capacities to manage complex delivery requirements).\n\n\nConclusions\n\nOur conclusions can be simply stated:\n\nadopting a societal perspective is not a context-free requirement,\n\nbeing explicit about the perspective is a context-free requirement,\n\nchoice of perspective is always and everywhere a matter for the study sponsors to determine together with any stakeholders they select,\n\nif the sponsors of a study intend it to inform policy decisions (public or otherwise) the decision makers’ perspective should be adopted (along with others deemed to be relevant by the sponsors),\n\nthe role of economists, epidemiologists and the like is to advise, assist, point out likely consequences, and to seek out, adduce, model and interpret evidence,\n\nthe role of economists, epidemiologists and the like is not to engage in advocacy for specific context-dependent perspectives, societal or otherwise.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by a grant from the Bill and Melinda Gates Foundation [OPP1134345] and the UK’s Department for International Development. Yot Teerawattananon holds the Thailand Research Fund Senior Research Scholar grant for Health Technology Assessment for Supporting Universal Health Coverage (grant number RTA59800011).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBell CM, Urbach DR, Ray JG, et al.: Bias in published cost effectiveness studies: systematic review. BMJ. 2006; 332(7543): 699–703. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPhilips Z, Ginnelly L, Sculpher M, et al.: Review of guidelines for good practice in decision-analytic modelling in health technology assessment. Health Technol Assess. 2004; 8(36): iii–iv, ix–xi, 1–158. PubMed Abstract \| Publisher Full Text\n\nOffice of Technology Assessment: The Implications of Cost-Effectiveness Analysis of Medical Technology. Washington, DC: Congress of the United States; 1980. 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PubMed Abstract \| Publisher Full Text\n\nReinhardt UE: Can efficiency in health care be left to the market? J Health Polit Policy Law. 2001; 26(5): 967–92. PubMed Abstract \| Publisher Full Text\n\nLeonard K, Mliga G, Mariam DH: Bypassing health centres in Tanzania: revealed preferences for quality. J Afr Econ. 2002; 11(4): 441–71. Publisher Full Text\n\nRieskamp J, Busemeyer JR, Mellers BA: Extending the bounds of rationality: evidence and theories of preferential choice. J Econ Lit. 2006; 44(3): 631–61. Publisher Full Text\n\nGrisso T, Applebaum PS: Assessing Competence to Consent to Treatment. New York: Oxford University Press; 1998. Reference Source\n\nSmith RD, Coast J: Controlling antimicrobial resistance: a proposed transferable permit market. Health Policy. 1998; 43(3): 219–32. PubMed Abstract \| Publisher Full Text\n\nCulyer AJ: The Political Economy of Social Policy. Oxford: Martin Robertson; 1983; 29–34." }	[ { "id": "29941", "date": "24 Jan 2018", "name": "David Tordrup", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a historical review of the origin of \"perspective\" in economic evaluation, and proceed to critique the standardisation of perspective, proposing that it is inherently context-specific. The paper is interesting and well written. I would make the following points:\nIf we accept that guidelines for EE written by e.g. Drummond et al. are intended for evaluation of healthcare interventions, with the decision problem being to adopt or not adopt a new technology, and that these guidelines are at least implicitly developed with the UK context in mind, this means that - yes - the guidelines are intended to generalise away from a specific context (the technology) - but - they also generalise within a specific context (the health system). The same can be said for methods guidance published by NICE. So there are situations where the end users of evidence are issuing guidance on the approach to EE generation, to determine best practices and what evidence is acceptable to the decision problem. The authors concede this, I believe, when they write in conclusion \"if the sponsors of a study intend it to inform policy decisions (public or otherwise) the decision makers’ perspectives should be adopted (along with others deemed to be relevant by the sponsors)\". This should mean that not all standardisation of perspective is necessarily bad.\n\nFurther to point 1, if the authors believe that each EE study intended to inform a public healthcare payer perspective should have its perspective built from scratch, how would the consumer of the evidence (the payer) avoid \"cherry picking\" by the sponsor (a manufacturer) of the most economically advantageous outcomes of the new technology under consideration? And if the perspective should not be built from scratch, how is that different from accepting to have some form of standardised guidance on perspective?\n\nThe authors also note capacity as a constraint in LMIC's, it seems point 2 would pose an even more substantial risk of \"evidence capture\" in such settings, where the majority of resource and technical capacity could easily be on the sponsor side, and not the evidence user side. In such cases, the capacity of the evidence user to articulate their needs could be severely limited.\n\nSo, while I fully agree that the choice of perspective should be determined by the user of the evidence, I think the authors should revisit their arguments against guidelines on perspective with the points above in mind. Are there cases where it is desirable to have standardised perspective guidance? What are the situations where it is undesirable - presumably where it is simply the result of asserting academic authority or other vested interest?\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [ { "c_id": "3535", "date": "22 Mar 2018", "name": "Benjarin Santatiwongchai", "role": "Author Response", "response": "In response to David Tordrup, we should emphasise that we neither said nor implied that a “standard” perspective is ”bad”. The question is: who chooses the perspective? NICE, CADTH and HITAP each has a standard perspective that is fit for its local purpose and chosen by those accountable for making decisions or recommendations in that context. Each of the authors of this paper has been both analyst and decision maker (the latter as the employee or non-executive officer of an HTA agency}. Our accountabilities in each role are not the same. In particular, our skills as scientists do not qualify us to make value judgments on behalf of society. At the most, the kind of value judgments we are entitled to make are value judgments about the quality of the science, which are certainly judgments of value, whether the science is good science, but they are not judgments about what is good for society. What qualified us as makers of social value judgments was our appointment through a legitimate process.Our view on perspective does not involve every study building a perspective from scratch. On the contrary, there is much to be said for establishing agencies like NICE etc. which are competent to set standards appropriate to their local context. In many cases analysts might usefully be employed in helping to build such a standard “from scratch” but, once set up, the work becomes one of maintenance as new techniques of analysis emerge, or new data, or new criteria are suggested. As far as any specific study is concerned, its authors should follow the guidance as set out by decision makers or else guess as best they may what the perspective might be of those whose decisions they seek to influence." } ] }, { "id": "29943", "date": "24 Jan 2018", "name": "Alexander Winch", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the topic of the opinion article discussed accurately in the context of the current literature?\nThis is a welcome and much needed addition to the dialogue on improving the quality of conducting and reporting economic evaluations and HTA internationally.\n\nWhilst the linkage is made well between the need for an explicit perspective as part of a robust high quality economic evaluation, one opportunity could be to explicitly state the importance of a well defined and robust decision problem as a fundamental to being able to define an appropriate perspective for an EE. Whilst this may be clear for those familiar with the guidelines, however the intended audience may not be familiar with the concept of scoped decision problems.\n\nThe section on the history of perspectives in methodological guidelines for conduction EE covers well the main evolution in guidelines. One potential enhancement could be through a discussion on why it took 20+ years for the perspective of an EE to be included explicitly as a characteristic in guidelines.\n\nThe article is clear on the justification for an explicit perspective, and why broader is not always better when it comes to 'societal perspective'. However, one opportunity for the article could be to look at the misuse or misunderstanding of concepts and language between policy makers and the technical EE analysts when it comes to the perspective on an evaluation. For example, policy decision makers may be supportive of using a 'societal perspective' for an economic evaluation in a context for which this may not be appropriate, yet actually be conflating this concept of the 'societal perspective' with undertaking an inclusive, stakeholder engagement strategy in the health decision making process. This conflation, is similar to the use by policy decision makers of terms such as 'cost-effective' of an intervention, even when this is not directly linked to the concept of opportunity cost, or the use of a context specific CE threshold, creating as a weird consequence the dichotomy of something being deemed 'cost-effective' yet 'unaffordable'.\n\nAre all factual statements correct and adequately supported by citations?\nAll factual statements are correct and supported by citations.\n\nParagraph 7 could strengthen the assertion that ‘Some LMICs, where out-of-pocket costs are significant select a societal perspective though very few LMICs have a track record of using EE systematically’ through references to LMICs with high OOPs, or LMICs track record of EE use in decision making\n\nAre arguments sufficiently supported by evidence from the published literature?\nAll arguments made in the article are supported by evidence from the published literature.\n\nIn paragraph 3, the assertion that 'a good study will be, therefore, explicit about its perspective’ could be enhanced through using terms such as 'high quality', 'economically rigorous' or 'comprehensive' as this can then be referenced back to the principles of the iDSI reference case.\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments?\nThe conclusions drawn are balanced and justified on the basis of presented arguments. Dependent on the intended audience for the article, the language could be simplified from 'context-free' and 'context-dependent' requirements, and instead restructure the conclusions for both the EE analyst (e.g. 1. advise, assist and point out likely consequences to decision maker 2) Not to engage in advocacy of any single perspective), and conclusions for the policy decision maker (1. adopt an explicit perspective in an EE 2. Be explicit in the reasons behind the choice of the perspective etc..)\n\nMinor additional comments:\n\nTable 2 displays payer, health care sector, societal as discrete categories – Does this double count those countries where multiple perspectives may be recommended in guidelines? Or are they given one discrete category? – For clarity it might be good to include a 'multiple' category to the table\n\nFor standardization and clarity, the language under column 'perspective' in Table 1 could be greater aligned. E.g: ‘Costs: Health care payer (government + patients); outcomes: society’ ‘Health care sector….’ ‘In the reference case, the perspective should be that of the publicly funded health care payer.’ ‘Health technology assessment (HTA) in Indonesia is expected to use societal perspective.’\n\nLast bullet point on conclusions is repeated\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3534", "date": "22 Mar 2018", "name": "Benjarin Santatiwongchai", "role": "Author Response", "response": "The authors have much sympathy with Alex Winch’s comment on the ambiguity of language and the very real possibility that the understanding of decision makers and technical experts may be significantly divergent. This is probably something that can only be addressed through training (especially the training of health economists) and through careful explaining and re-explaining. It is not helped by devising new names for economic evaluation (currently running at around 24 near synonyms) or by inconsistent use of definitions even amongst economists (a spectacular demonstration of such disagreement about “opportunity cost” is Ferraro and Taylor 2005, but much the same embarrassing variation exists concerning “comparative and absolute advantage”, “cost-effectiveness”, “efficiency” or “equity”).Ferraro PJ, Taylor LO (2005) Do economists recognize an opportunity cost when they see one? A dismal performance from the dismal science, Contributions in Economic Analysis & Policy, 4. doi:10.2202/1538-0645.1469." } ] }, { "id": "30070", "date": "07 Mar 2018", "name": "Carol Levin", "expertise": [ "Reviewer Expertise Global health economics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAs a practitioner who conducts costs and cost-effectiveness analysis in low and middle-income countries, this was a useful paper and reminder to consider context and include stakeholder input early in the process as best practice. I appreciated the history of perspective and the clear conclusions simply stated. I have replied partly to two of reviewer questions because I read this opinion paper through the lens of a practitioner working only in a global health context, with familiarity of the published papers across a number of global health conditions.\n\nAs I read the paper, I wondered who the audience is for this paper? Is it researchers in high-income countries? Is it intended for researchers in global health economics? Is the purpose of the paper to counter recent recommendations of the second panel on cost-effectiveness or the second “Washington Panel”?\n\nI wasn’t quite sure what the problem being addressed is and who the intended audience for this paper was, since it combined a mix of practical points, theory and recommendations.\n\nRecommendation: In the introduction, can the authors add a problem statement? And then explicitly indicate if these recommendations are for researchers in high-income countries or global health settings or both.\n\nAlso, as a practitioner, my experience is that analysts, conducting global health economic evaluations, more often than not, use common sense and already use a provider or (provider and societal perspective) in the majority of published economic evaluations in global health (whether for evaluating health technology, vaccines, diagnostics, medicines, delivery strategies). For instance, I selected two annexes available through the Disease Control Priorities Project for Reproductive Maternal and Child Health CEA studies and for Cardiovascular CEA studies and found that out of 295 articles, 22% were conducted from a societal perspective and 52% were from a provider perspective and 15% were from both. For CVD prevention or treatment CEA studies, 27% were from a societal perspective and 68% from a provider perspective.\n\nThis may not be the same as more deliberately using context specific perspective. So this either means that researchers conducting global health economic evaluations are already pragmatic, engaging stakeholders to select a context specific perspective, or that they are not following recommended guidelines. The authors note that only a third of the guidelines from both HIC and LMIC recommend a societal perspective. That certainly makes sense to me, for LMIC where economic evaluations are often conducted to introduce a new health technology or delivery strategy, where the status quo is nothing (or very low coverage of existing technologies or services).\n\nAs the authors nicely present, EE was method first developed for use in high-income countries, with a very specific objective in mind. They were not developed with global health interventions and strained governance and health systems in mind. However, as interest and capacity grows in LMIC to conduct EE, guidelines are extremely useful for researchers at nascent stages of learning a new method. And these guidelines typically get adapted initially and over time to better suit applications in global health, through disease specific recommendations/guidance/guidelines promoted by WHO or other multi-lateral or global health organizations.\n\nOn page 4, the authors note that there can be no presumption that one size fits all, and states we should not encourage a similar intensity to enter an ‘authoritative methodological guide to best practice in EE’. I’m not sure that analysts DO presume a one size fits all. This is very clear from any review of a set of CEA studies that look at exactly the same two alternative strategies to address the same health condition. Comparability across many studies is difficult. I’m also not sure whom the authors are referring to and if there is a problem among researchers conducting global health EE with respect following authoritative guidelines (There are many problems in standard methods and reporting across studies—and these do cause a problem for using resulting study results—suggesting that guidelines are important to improve quality and comparability of studies—even when appearing authoritative—not sure what is wrong with that when talking about methods). When the guidance is not feasible or even does not make sense (i.e. using a societal perspective), it does seem that these recommendations are simply not followed. This may explain why provider perspective is used more often than provider perspective in EEs in LMIC.\n\nRecommendation: Is it possible to take into consideration what is done in practice and acknowledge that practice already is very different from the guidance—certainly in LMIC?\n\nRecommendation: On page 3, column 1, par 2, suggest indicating that guides to best practice can be traced back at least as far as 1980 developed for use in high income countries to inform government allocation of resources (or something to this effect). Very important to note these guidelines were NOT initially developed for LMIC, but adapted more recently for that purpose.\n\nQuality and comparability in economic evaluations is challenging. In the introduction, the authors recognize the need to set international quality standards, but I take some issue in calling those that follow the standards falling into a “trap of small-town thinking”. Perhaps this is not what the authors meant, but I’m not sure that it is parochialism at work, but rather a desire to generate comparable results, using economic theory as a foundation for the recommendation. Perhaps it is this underlying theory that the authors take issue with.\n\nThe authors understand the history and economic theory underlying the recommendation of a societal perspective much better than I, but it is unclear to me what the economic debate is and between who? They allocate a section in the paper to a theoretical discussion on the issue of individual expressions of value and collective ones. While I found this interesting, it seemed somewhat out of place to me, if this paper’s audience is practitioners in low and middle-income countries, doing their best to figure out the best way to conduct economic evaluations.\n\nRecommendation: What is the consequence of wrongly using a societal perspective? Can the authors more simply explain this to readers in the introduction or somewhere in the paper why a societal perspective may not allocate resource efficiently and equitably?\n\nThis gets me to my final comment on the paper, which it is a mix of a thought paper and a recommendation or guidance paper. I found myself wondering about the ‘how’ on page 9 when authors suggest engaging a long list of stakeholders in determining the perspective of the EE analysis. I agree with this, but can the authors refer the reader or add information on how to do this? This becomes particularly important for academics that are conducting EE in LMIC without direct access to stakeholders. This is often the case for researchers conducting EE for a specific health technology (vaccines, drugs, diagnostic, mhealth application,) that is under development. In addition, even when new services or technologies are funded and ready for introduction in LMIC, demand comes from the donor, researcher or NGO, not the in-country stakeholders that will ultimately introduce and fund the health intervention. However, I fully agree, when stakeholders are involved, identifying who will USE the information, and engaging them early makes it much easier to determine the objective and perspective of the study.\n\nMinor comment\nAbstract 1. Suggest that the authors provide a main recommendation in the abstract. Or end the abstract with a more positive statement stating that economic evaluation becomes more relevant when the perspective is sensitive to context and appropriate for the objective of the analysis.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3533", "date": "22 Mar 2018", "name": "Benjarin Santatiwongchai", "role": "Author Response", "response": "The authors are grateful to the reviewers for the evident care they have taken in reviewing our paper.Carol Levin requests a problem statement and greater clarity as to its intended readership. We thought we had stated the “problem” clearly in the Introduction, namely that what we called “small-town thinking” imposes arbitrary limits on the perspective from which economic evaluations are conducted. Furthermore, we do not regard it as the business of analysts (when acting as analysts) to assert appropriate perspectives. It follows that, since this is a point about scientific method, our article is addressed at one level to methodologists, textbook writers and others who seek to provide training materials and set standards of good practice for economic evaluations. Since good practice is something that practitioners ought to follow, practitioners need to be alerted to the possibility that not all expert recommendations on good practice may be sound. While this is a perfectly general point, which requires us all to be critically alert to the possibility of poor guidance, we quite specifically aimed a criticism at the common recommendation that studies be conducted from the “societal” perspective. We offered a little history of this approach, which has begun to assume the status of a common standard. The main reason for our view is that to select a perspective (any perspective) is to make a social value judgment about what “counts” in designing and implementing public policy, including health policy. The perspective defines the benefits, costs and harms that are to be considered as well as the ways in which they are to be brought into a single judgment about the eligibility of clinical procedures for inclusion in an insurance (public or private) scheme. Analysts are not empowered (qua analysts) to make these judgments. Those who are empowered include the managers of private health insurance schemes (typically accountable to a board, to trustees, or to shareholders) and the managers of public schemes, usually accountable to a minister and through the minister to an electorate. These people are commonly referred to as “decision makers”. This accountability does not apply to analysts – unless of course they are themselves also managers, trustees, etc. Accordingly, good practice for analysts is to assist decision makers in selecting a perspective that is defensible and in accordance with the social and political values the decision makers in question are expected to live up to.One way in which this can be done is to have the decision makers endorse a formal methodological approach that should characterise all the analysis commissioned for that specific jurisdiction. This is very common amongst jurisdictions that have set up specialist agencies to make decisions (like NICE) or to make recommendations (like HITAP or CADTH). We observed in our review that the societal perspective does not usually characterise these guides to method, or “reference cases”. Nor are they by any means identical, despite having been devised with assistance of evidently competent economic analysts. This should not be a matter for regret. Rather, the presumption is that each has been designed for the particular circumstances of the jurisdiction to which it applies. Amongst other implications is that academics conducting analyses “without direct access to stakeholders”, as Dr Levin puts it, are at great risk of producing recommendations that are irrelevant in any specific context. Good practice does not invariably require huge amounts of interaction with stakeholders but when the client for analysis has made no statement about perspective, and stakeholders are beyond reach, good practice is most likely to be “no practice”. For whom, one might ask, might such an uninformed analysis be intended?" } ] } ]	1	https://f1000research.com/articles/7-72
https://f1000research.com/articles/7-71/v1	17 Jan 18	{ "type": "Research Article", "title": "A TALE of shrimps: Genome-wide survey of homeobox genes in 120 species from diverse crustacean taxa", "authors": [ "Wai Hoong Chang", "Alvina G. Lai", "Wai Hoong Chang" ], "abstract": "The homeodomain-containing proteins are an important group of transcription factors found in most eukaryotes including animals, plants and fungi. Homeobox genes are responsible for a wide range of critical developmental and physiological processes, ranging from embryonic development, innate immune homeostasis to whole-body regeneration. With continued fascination on this key class of proteins by developmental and evolutionary biologists, multiple efforts have thus far focused on the identification and characterization of homeobox orthologs from key model organisms in attempts to infer their evolutionary origin and how this underpins the evolution of complex body plans. Despite their importance, the genetic complement of homeobox genes has yet been described in one of the most valuable groups of animals representing economically important food crops. With crustacean aquaculture being a growing industry worldwide, it is clear that systematic and cross-species identification of crustacean homeobox orthologs is necessary in order to harness this genetic circuitry for the improvement of aquaculture sustainability. Using publicly available transcriptome data sets, we identified a total of 4183 putative homeobox genes from 120 crustacean species that include food crop species, such as lobsters, shrimps, crayfish and crabs. Additionally, we identified 717 homeobox orthologs from 6 other non-crustacean arthropods, which include the scorpion, deer tick, mosquitoes and centipede. This high confidence set of homeobox genes will now serve as a key resource to the broader community for future functional and comparative genomics studies.", "keywords": [ "Crustacean", "homeobox", "TALE", "comparative genomics", "arthropod", "homeodomain" ], "content": "Introduction\n\nAs one of the fastest growing industries, the seafood trade is dominated by fishing and farming of crustaceans, with annual sales exceeding $40 billion (Stentiford et al., 2012). Crustacean aquaculture is multi-faceted, not only contributing to the ever-increasing demands by international markets, but is also directly linked to the socio-economic aspects of many developing nations through the creation of jobs and infrastructure. Aquaculture practices have intensified in recent years to cope with the demand. Yet, many are not sustainable since the increased densities of farmed shrimps often serve as hotbeds for pathogens if left unabated, causing infectious diseases and the devastation of cultures resulting in massive financial losses. As a result, regulations associated with aquaculture diseases are being enforced with emphasis placed on preventative measures, e.g. enhancement of broodstock and research aiming to further our understanding on crustacean development and ways to utilize the innate ability of crustaceans to combat pathogens (Lai & Aboobaker, 2017; Stentiford et al., 2012).\n\nSeveral conserved molecular genetic circuitries are well-known for regulating many aspects of development and innate immune homeostasis. One prominent example would be homeobox genes, a family of transcription factors defined by the presence of a homeodomain (Holland, 2013). As one of the most important master controls in development, some headway has already been made in understanding the involvement of homeobox genes in innate immunity; Caudal in Drosophila melanogaster is implicated in commensal-gut mutualism (Ryu et al., 2004; Ryu et al., 2008). Given their importance, major efforts have thus far focused on characterization of homeobox genes in well-known model organisms such as humans (Garcia-Fernàndez, 2005; Holland et al., 2007), Caenorhabditis elegans (Bürglin, 1997), D. melanogaster (Mukherjee & Bürglin, 2007), planarians (Currie et al., 2016; Felix & Aboobaker, 2010; Garcia-Fernandez et al., 1991), amphioxus (Luke et al., 2003), teleost fish (Mulley et al., 2006) and many more. Although homeobox orthologs have been previously studied in the crustacean Parhyale hawaiensis (Kao et al., 2016), systematic and cross-species characterization of this gene family across the broader Crustacea with focus on food crop species is currently lacking. A better understanding of homeobox genes in crustaceans is therefore required to address this major shortfall, leading us to our present work.\n\n\nMethods\n\nWe retrieved complete transcriptome data sets for 120 crustacean species available at the time of manuscript preparation from the European Nucleotide Archive. Six non-crustacean arthropod proteomes were retrieved from Uniprot. A complete list of accessions used in this study is provided in Supplementary Table 1. We retrieved a list of query sequences used in subsequent homology searches from Uniprot and GenBank.\n\nBased on a previously published workflow (Lai & Aboobaker, 2017), we used multiple Basic Local Alignment Search Tool (BLAST)-based approaches, such as BLASTp and tBLASTn to identify genes with homeodomain sequences. The BLAST results were filtered by e-value of < 10-6, best reciprocal BLAST hits against the GenBank non-redundant (nr) database and redundant contigs having at least 95% identity were collapsed using CD-HIT. We then utilized HMMER (version 3.1) employing hidden Markov models (HMM) profiles (Finn et al., 2011) to scan for the presence of Pfam homeodomains (Bateman et al., 2004) on the best reciprocal nr BLAST hits, to compile a final non-redundant set of crustacean and arthropod homeobox gene orthologs (Dataset 1).\n\nMultiple sequence alignment of homeodomain sequences was performed using MAFFT (version 7) (Katoh et al., 2009). Phylogenetic tree was built from the MAFFT alignment using RAxML WAG + G model to generate a best-scoring maximum likelihood tree (Stamatakis, 2014). Geneious (version 7) was used to generate a graphical representation of Newick tree (Kearse et al., 2012).\n\n\nResults and discussion\n\nWith the recent availability of a large number of transcriptome data sets, we perform an extensive search for homeobox genes from 120 crustacean species. We focus on species represented across the broader Crustacea sampling from three main crustacean classes, Malacostraca, Branchiopoda and Copepoda, with focus on key food crop species from the order Decapoda (Supplementary Table 1). Using BLAST-based approaches and profile HMM (Bateman et al., 2004; Finn et al., 2011; Finn et al., 2015) for homology searches, we conservatively identified 4183 transcripts with homeodomain sequences from crustaceans (Figure 1; Dataset 1). Additionally, we included six non-crustacean arthropod species in our search and from these species, we identified 717 homeobox orthologs (Figure 1; Dataset 1).\n\n(A) Number of homeobox gene orthologs identified in each species are depicted as boxplots, indicating the median and quartiles. Violin plots underlying the boxplots illustrate sample distribution across different crustacean taxa and kernel probability density (width of the shaded areas represent the proportion of data located in these areas). The homeobox gene orthologs from six non-crustacean species within Arthropoda (others) are also shown. (B) Bar charts illustrating the number of homeobox gene orthologs in crustaceans from Decapoda, Branchiopoda and Copepoda along with six non-crustacean arthropods (others).\n\nConcerted efforts to establish evolutionary classification of homeobox genes have resulted in 11 recognised classes (Edvardsen et al., 2005; Holland et al., 2007; Ryan et al., 2006; Zhong et al., 2008; Zhong & Holland, 2011). The Three-Amino acid-Loop Extension (TALE) superclass within the group of homeobox genes is characterized by three additional residues between alpha helices 1 and 2 of the homeodomain (Bertolino et al., 1995). TALE class homeodomain proteins are further divided into 6 subclasses, Meis, Pknox, Pbc, Irx, Mkx and Tgif characterized by distinct motifs beyond the homeodomain (Bürglin, 1997; Bürglin, 2005; Holland et al., 2007; Mukherjee & Bürglin, 2007). We have classified a total of 165 TALE class orthologs from 15 decapod crustacean species (Figure 2). These genes form distinct phylogenetic grouping, which allows confident assignment of decapod TALE class orthologs into 6 sub-families (Figure 2). Importantly, the tree topology of crustacean TALE class orthologs recapitulated observations from a previous study (Holland et al., 2007).\n\nThe tree was constructed using the maximum-likelihood method from an amino acid multiple sequence alignment, which include TALE class genes from other species (Zhong et al., 2008 and Zhong & Holland, 2011). TALE orthologs representing 6 subclasses are colour-coded. The node labels of each taxon are marked with distinctive colors denoted in the figure inset. Bootstrap support values (n=1000) are denoted as branch labels.\n\n\nConclusion\n\nWe identified 4900 homeodomain transcripts from 120 crustaceans and 6 non-crustacean arthropod species. Although this data set is non-exhaustive – transcriptomes contain only genes expressed at the point of sample collection – it will now serve as a key resource for future functional studies in the context of crustacean aquaculture. Beyond crustaceans, this work is widely applicable to studies on homeobox genes from other animals and will facilitate evolutionary and comparative genomics investigations.\n\n\nData availability\n\nDataset 1: List of Pfam annotated homeobox genes and associated e-values in crustaceans and other arthropods. DOI, 10.5256/f1000research.13636.d190417 (Chang & Lai, 2018).\n\nDataset 2: Fasta file for homeobox gene sequences in crustaceans and other arthropods. DOI, 10.5256/f1000research.13636.d190418 (Chang & Lai, 2018).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the EMBO Fellowship and the Human Frontier Science Program Fellowship to AGL.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Table 1: List of accession numbers for species used in this study.\n\nClick here to access the data.\n\n\nReferences\n\nBateman A, Coin L, Durbin R, et al.: The Pfam protein families database. Nucleic Acids Res. 2004; 32(Database issue): D138–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBertolino E, Reimund B, Wildt-Perinic D, et al.: A novel homeobox protein which recognizes a TGT core and functionally interferes with a retinoid-responsive motif. J Biol Chem. 1995; 270(52): 31178–31188. PubMed Abstract \| Publisher Full Text\n\nBürglin TR: Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic Acids Res. 1997; 25(21): 4173–4180. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBürglin TR: Homeodomain proteins. Rev Cell Biol Mol Med. 2005.\n\nChang WH, Lai AG: Dataset 1 in: A TALE of shrimps: Genome-wide survey of homeobox genes in 120 species from diverse crustacean taxa. F1000Research. 2018. Data Source\n\nChang WH, Lai AG: Dataset 2 in: A TALE of shrimps: Genome-wide survey of homeobox genes in 120 species from diverse crustacean taxa. F1000Research. 2018. Data Source\n\nCurrie KW, Brown DD, Zhu S, et al.: HOX gene complement and expression in the planarian Schmidtea mediterranea. Evodevo 2016; 7: 7. 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PubMed Abstract \| Publisher Full Text\n\nGarcia-Fernandez J, Baguñà J, Saló E: Planarian homeobox genes: cloning, sequence analysis, and expression. Proc Natl Acad Sci U S A. 1991; 88(16): 7338–7342. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHolland PW: Evolution of homeobox genes. Wiley Interdiscip Rev Dev Biol. 2013; 2(1): 31–45. PubMed Abstract \| Publisher Full Text\n\nHolland PW, Booth HA, Bruford EA: Classification and nomenclature of all human homeobox genes. BMC Biol. 2007; 5: 47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKao D, Lai AG, Stamataki E, et al.: The genome of the crustacean Parhyale hawaiensis, a model for animal development, regeneration, immunity and lignocellulose digestion. eLife. 2016; 5: pii: e20062. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKatoh K, Asimenos G, Toh H: Multiple alignment of DNA sequences with MAFFT. Methods Mol Biol. 2009; 537: 39–64. PubMed Abstract \| Publisher Full Text\n\nKearse M, Moir R, Wilson A, et al.: Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 2012; 28(12): 1647–1649. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLai AG, Aboobaker AA: Comparative genomic analysis of innate immunity reveals novel and conserved components in crustacean food crop species. BMC Genomics. 2017; 18(1): 389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuke GN, Castro LF, McLay K, et al.: Dispersal of NK homeobox gene clusters in amphioxus and humans. Proc Natl Acad Sci U S A. 2003; 100(9): 5292–5295. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMukherjee K, Bürglin TR: Comprehensive analysis of animal TALE homeobox genes: new conserved motifs and cases of accelerated evolution. J Mol Evol. 2007; 65(2): 137–153. PubMed Abstract \| Publisher Full Text\n\nMulley JF, Chiu CH, Holland PW: Breakup of a homeobox cluster after genome duplication in teleosts. Proc Natl Acad Sci U S A. 2006; 103(27): 10369–10372. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyan JF, Burton PM, Mazza ME, et al.: The cnidarian-bilaterian ancestor possessed at least 56 homeoboxes: evidence from the starlet sea anemone, Nematostella vectensis. Genome Biol. 2006; 7(7): R64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRyu JH, Kim SH, Lee HY, et al.: Innate immune homeostasis by the homeobox gene caudal and commensal-gut mutualism in Drosophila. Science. 2008; 319(5864): 777–782. PubMed Abstract \| Publisher Full Text\n\nRyu JH, Nam KB, Oh CT, et al.: The homeobox gene Caudal regulates constitutive local expression of antimicrobial peptide genes in Drosophila epithelia. Mol Cell Biol. 2004; 24(1): 172–185. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStamatakis A: RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics. 2014; 30(9): 1312–1313. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStentiford GD, Neil DM, Peeler EJ, et al.: Disease will limit future food supply from the global crustacean fishery and aquaculture sectors. J Invertebr Pathol. 2012; 110(2): 141–157. PubMed Abstract \| Publisher Full Text\n\nZhong YF, Butts T, Holland PW: HomeoDB: a database of homeobox gene diversity. Evol Dev. 2008; 10(5): 516–518. PubMed Abstract \| Publisher Full Text\n\nZhong YF, Holland PW: HomeoDB2: functional expansion of a comparative homeobox gene database for evolutionary developmental biology. Evol Dev. 2011; 13(6): 567–568. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "29917", "date": "14 Feb 2018", "name": "Ricardo M. Zayas", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe report by Chang and Lai provide an extensive dataset and phylogenetic analysis crustacean homeobox genes. This data will be useful to individuals interested in studying the evolution and function of homeobox genes in crustacea and other organisms. Overall, this manuscript is well written and appears to be technically sound. I only have some very minor comments the authors could address to improve clarity:\n1) In the abstract, the rationale for the work takes an intellectual leap: it is unclear how identification of homeobox genes will be useful for aquaculture sustainability. I think the authors provide some compelling reasons within the text of the manuscript.\n2) In the results and discussion there are a few instances where the authors should use the past tense.\n3) Page 3, last sentence: even though the authors referred to Peter Holland's work, they should be much more precise about the observations from a \"previous study\". The authors should expand what they mean.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "32836", "date": "13 Apr 2018", "name": "Nathan J Kenny", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis work makes a putative assessment of the overall homeodomain complements of the transcriptomes of a number of crustacean species. One class of homeodomain containing genes (TALE) from one order of crustaceans (Decapoda) is assessed in detail, but otherwise no attempt is made to categorise putative hits into gene families. The work is therefore preliminary in scope, and would benefit from the provision of even broadest-level classification of hits into appropriate classes/subclasses of gene, which should be fairly straightforward given the diagnostic residues used to categorise these classes.\nThe title as it stands is misleading - a genome wide survey is not made, and instead transcriptomic data is used, which will (by necessity) be gappy.\nA link is made between aquaculture, innate immunity and homeodomain-containing proteins, but this is very tenuous. Particularly, why the focus is on TALE class genes is unclear, if Caudal is the gene used as the exemplar for a link between these fields?\nThe methods section needs to be more precise. For example: \"such as BLASTp and tBLASTn to identify genes with homeodomain sequences\". What was done? How were protein sequences derived from nucleotide data for blastp searches? What sequences were used to search your datasets? (Perhaps add these to the sentence: \"list of query sequences used in subsequent homology searches from Uniprot and GenBank.\"). The latter is particularly important as more distant sequences may be missed.\nI have several questions about e values. -Dataset 1 contains several ID'd genes with higher E values than the stated cutoff (< 10-6). Is this deliberate? -The e-value of < 10-6 will also likely result in larger datasets returning more hits, purely as a consequence of how the E (expect) value is calculated. For example, the Daphnia magna transcriptome has 271,000 sequences, Triops 12,000. Therefore it is much more likely that sequences will make it through your annotation pathway in Daphnia magna rather than Triops. This will skew the results shown in Fig 1B. Is it possible to show that homeodomain genes are not artificially excluded, perhaps by giving the E values and best blast hits from \"next-best\" excluded sequences in your initial searches of small datasets, to prove no homeodomain sequences were artificially excluded? This is crucial, given the short length of the homeodomain, which will be the primary source of signal.\nWas HMMR really run on the best reciprocal nr hits, as is suggested by your phrasing? Or was it run on the transcriptome-derived data?\nFig 1A: Violin plots are not appropriate here. Look for instance at the Branchiopod data, where 3 points are used to infer this plot.\nThe results in the tree in Fig 2 seem to indicate that decapod crustaceans completely lack Mkx genes, and the presentation of this is disingenuous in text (note the paraphyly of known Mkx homologues with regard to the inferred crustacean Mkx). Instead, the crustacean Mkx seem to be Pbc?\n\"Importantly, the tree topology of crustacean TALE class orthologs recapitulated observations from a previous study (Holland et al., 2007).\" - this statement does not seem to be correct.\nThe homeodomain complements, especially of ANTP class genes, of several crustacean species have been described previously, but no attempt is made to place the results observed in the context of the annotated sets of other species. Could this be provided? Particularly, the utility of the re-assessment of non-crustacean datasets is unclear, as these resources have been annotated previously in more detail. Were additional homeodomain-containing genes found by this re-analysis? Or fewer?\nIn short, this work seems to be partially successful in its aims. With the addition of additional information about the identity of sequences, and the correction of the problems noted above, it will be a coherent addition to extant information on crustacean homeodomain-containing genes.\nMinor comments: $40 billion - which currency? USD?\nThere are several areas where the phrasing could be improved, e.g. -\"With continued fascination on this key class of proteins\"\nSometimes articles (a/the) are missing from the text, e.g. - \"Phylogenetic tree was built from\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "33514", "date": "23 May 2018", "name": "Colleen Doherty", "expertise": [ "Reviewer Expertise Transcriptional networks" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Chang and Lai identify sequences of the homeobox genes in crustaceans from transcriptional data. For TALE family members, they classify these orthologs into the six subfamilies. The introduction provides the justification for establishing this resource in these agriculturally important species. In total, this will be an important reference source for future work in understanding the transcriptional regulation of development in crustacean species. There are a few minor questions and suggestions the authors can address.\nThe details of the BLAST-based approaches and CD-Hit should be described in more detail. Were default settings applied? Perhaps adding an additional supplemental file or link with the commands would clarify this to facilitate replication in other species.\n\nThe authors have the opportunity to address how effective their approach is and potential places for improvement. For example, how do the datasets derived from proteomes compare to that from transcriptomes? Is there an overlapping dataset where the two approaches could be compared? Also, how does the identification of homeobox genes using this approach in Drosophila compare to the number annotated in this well-studied species? If all are identified this would suggest that this approach may be sufficient if some are missing it suggests that this conservative starting point could be enhanced in the future by additional refinement.\n\nMinor comments: Introduction, second paragraph, description of homeobox identification in other species. Either provide references for “and many more” or omit. As is, this phrase is vague and doesn’t add constructively to the introduction.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-71
https://f1000research.com/articles/6-1808/v1	06 Oct 17	{ "type": "Research Article", "title": "The peer review process for awarding funds to international science research consortia: a qualitative developmental evaluation", "authors": [ "Stefanie Gregorius", "Laura Dean", "Donald C Cole", "Imelda Bates", "Laura Dean", "Donald C Cole", "Imelda Bates" ], "abstract": "Background: Evaluating applications for multi-national, multi-disciplinary, dual-purpose research consortia is highly complex. There has been little research on the peer review process for evaluating grant applications and almost none on how applications for multi-national consortia are reviewed. Overseas development investments are increasingly being channelled into international science consortia to generate high-quality research while simultaneously strengthening multi-disciplinary research capacity. We need a better understanding of how such decisions are made and their effectiveness. Methods: An award-making institution planned to fund 10 UK-Africa research consortia. Over two annual rounds, 34 out of 78 eligible applications were shortlisted and reviewed by at least five external reviewers before final selections were made by a face-to-face panel. We used an innovative approach involving structured, overt observations of award-making panel meetings and semi-structured interviews with panel members to explore how assessment criteria concerning research quality and capacity strengthening were applied during the peer review process. Data were coded and analysed using pre-designed matrices which incorporated categories relating to the assessment criteria. Results: In general the process was rigorous and well-managed. However, lack of clarity about differential weighting of criteria and variations in the panel’s understanding of research capacity strengthening resulted in some inconsistencies in use of the assessment criteria. Using the same panel for both rounds had advantages, in that during the second round consensus was achieved more quickly and the panel had increased focus on development aspects. Conclusion: Grant assessment panels for such complex research applications need to have topic- and context-specific expertise. They must also understand research capacity issues and have a flexible but equitable and transparent approach. This study has developed and tested an approach for evaluating the operation of such panels and has generated lessons that can promote coherence and transparency among grant-makers and ultimately make the award-making process more effective.", "keywords": [ "Peer review", "review panels", "grant applications", "research consortia", "research capacity", "Africa" ], "content": "Introduction\n\nThe use of peer review by expert panels is a well established method for assessing scientific research and for evaluating grant applications (Abdoul et al., 2012; Coryn et al., 2007; Lawrenz et al., 2012; Wooten et al., 2014). Most of the literature on peer review focusses on how to ensure transparency and reliability in editorial peer review. However, except for Lamont’s (2009) in-depth work, little research has been conducted into the peer review process of grant applications; the few studies that have been conducted focused on individual research projects (Abdoul et al., 2012) and national research collaborations (Klein & Olbrecht, 2011).\n\nOverseas aid investments in science and technology are increasingly being channelled into international consortia models (El Ansari et al., 2007; UKCDS, 2015). Such research consortia usually comprise research institutions in high-income countries (HIC) and low- and middle-income countries (LMIC). Such consortia generally aim to generate innovative science through world class research and to strengthen research capacity at the individual, institutional and national/international level. For the HIC country partners, these collaborations provide a rich experience and understanding of working in developing countries and opportunities to adapt innovations for different contexts. The LMIC institutions benefit from exposure to a diversity of world class equipment and facilities that help strengthen individual and institutional research capacity (Dean et al., 2015; Syed et al., 2012).\n\nMuch of the literature on peer review has focussed on review of articles for publication. Peer review by panels evaluating research applications has received much less attention (Klein & Olbrecht, 2011) and we could find no publications about the peer review process for multi-national science consortia. There is therefore almost no evidence to guide good practice for peer review of these complex applications. In order to select consortia for funding, peer review panels have to assess consortia’s potential for achieving the dual aims of generating high quality science and strengthening LMIC research capacity at all three levels (i.e. individual, institutional, societal). These aims are complex and interlinked, and so the review panel need to have topic- and context-specific expertise and a flexible but equitable approach (Wessely, 1998). Given the amount of money invested globally in trans-national research consortia, there is a pressing need to understand how funding decisions are made and to develop an evidence base that can help to promote coherence and transparency within and among grant-makers, to ultimately make the process more effective.\n\nThis article describes the use of qualitative research to explore the peer review process used for awarding grants to ten multi-national natural science research consortia. The ten consortia were funded through a UK grant-making institution over two annual rounds of applications (2014 and 2015). Each consortium consists of three research institutions in sub-Saharan Africa (SSA) and one in the United Kingdom (UK). Applications were restricted to three research priority areas: renewable energy, soil-related science and water and sanitation. In addition to generating high-quality research, a key goal of the programme was to strengthen the research capacity of universities and research institutions in SSA by strengthening research infrastructure and the development of sustainable research networks. This was to be achieved by establishing multidisciplinary partnerships between the UK and SSA, strengthening research training capacity in SSA and establishing a PhD scholarship scheme with shared UK-SSA supervision.\n\nThe study into the peer review process was conducted by the Capacity Research Unit (CRU) at the Liverpool School of Tropical Medicine (LSTM). CRU is an independent research team, external to the grant-making body. CRU’s role was to undertake a developmental evaluation of the programme, conduct research into the research capacity strengthening processes of the programme and generate learning that could be used to improve the programme in (near to) real-time. Developmental evaluation (Patton, 2011) is an evaluation approach that helps to introduce change within uncertain and complex environments. It provides feedback to programme staff to inform a quality improvement loop. Consequently, this paper describes the process for selecting consortia in round one and using learning from this round to adjust the round two selection process. The research focused on exploring how closely the peer review selection process matched the overall goal of the programme, how reviewers used the assessment criteria and how final funding decisions were reached.\n\n\nMethods\n\nIn the absence of validated, published methods for research in evaluating peer review of multi-national research consortia, we applied relevant, well-established, qualitative research methods to collect data at three stages of the selection process: pre-award document review, observation of the award-making panel meetings and post-award interviews with panel members. The selection panel consisted of 20 peer reviewers. The face to face meetings of the panel were attended in person by 17 (round one) and 16 (round two) reviewers. Panel members unable to attend the face to face meeting provided written comments. The panel comprised three women and 14 men (round one) and three women and 13 men (round two). Three reviewers for each round were from Africa and the rest were from the UK or European Union. Their expertise covered water and sanitation (two per round), soil science (two per round), renewable energy (seven per round) and other relevant disciplines (5–6 per round).\n\nStructured overt observations of the two panel meetings were carried out to systematically document the process (Supplementary file 1 and Supplementary file 2) and semi-structured interviews (Supplementary file 3) were conducted with purposively selected panel members after the meetings. The interviews were designed to gain in-depth understanding of how the assessment criteria were understood and used and perceptions about the overall selection process. They were also used to validate data obtained from observations of the selection panel meetings.\n\nAmong the 73 eligible applications (26 in round one, 47 in round two) submitted to the award-making institution, 34 were shortlisted for full review (15 in round one, 19 in round two): 6/6 (round one/round two) in renewable energy, 4/8 in soil-related science, and 5/5 in water and sanitation. We were not involved in the shortlisting process. Each eligible application was scored by three panel members who were assigned applications on the basis of their relevant expertise. Applications that met the assessment criteria and had highest scores were shortlisted. Each shortlisted application was reviewed by at least five external specialists and their comments taken into consideration during the final selection meeting.\n\nGuidance notes for panel members on the conduct of the face to face meeting and the assessment criteria, and summary details of the shortlisted applications for both rounds, were provided to us by the grant-awarding institution. We used a pre-designed matrix to extract information from the panel guidance notes about the assessment criteria against which applications were to be judged, the role of the panel members, the role of the panel chair and the panel code of conduct.\n\nOvert observation of the panel meetings involved the direct observation (Lamont, 2009; McNaughton et al., 2013) of the panel in their natural setting; as observer-researchers we did not participate in the process. The observations of each panel review meeting were conducted by three (round one) or two (round two) researchers. The researchers gave a brief presentation at the beginning of each meeting outlining the role of CRU within the programme and the purpose of their observations during the panel meetings. Panel members were given the opportunity to ask questions and the researchers gave an undertaking to maintain confidentiality and anonymity.\n\nAn iterative approach was used for the observation of the round one panel meeting to allow flexibility in the research approach. This was important as there was no published relevant research in this area and there were no appropriate data collection tools or findings that could be used or adapted. We used the assessment criteria in the panel guidance document to develop tools that enabled data to be collected against each criterion during observation of the selection panel meeting (see box 1 and box 3). In addition, the data collection for the observation of the panel meeting for the first round of shortlisted applications was partially influenced by a Swedish Research Council publication that broadly explored the modus operandi of its evaluation panels (Ahlquist et al., 2013). This document primarily influenced the development and content of observation matrices used for note-taking during the overt observation.\n\n\n\nPrimary considerations\n\nQuality of the research\n\nQuality of the research teams involved\n\nQuality of candidates for PhD scholarships (if already identified)\n\nQuality of the training/supervision plans for:\n\nPhD Students covered by scholarships\n\nWider training programme for other researchers and technicians\n\nAdditional considerations:\n\nIndication of host institutions’ capability and willingness to utilise the programme for the development of their own research strategy\n\nDiversity: good spread across the SSA region, including non-Anglophone countries\n\nSpread across the three priority research areas\n\nSupport for early to mid-career scientists\n\nSupport for female scientists\n\n\n\n1. Applications should initially be assessed based on scientific strengths by considering the following points:\n\n• Excellent quality of overall research programme\n\n• Strong individual research projects\n\n• Strong research background of scientists\n\n• Strong track record of host institutions\n\n• Excellent quality of proposed PhD research projects (including how well they integrate with overall research project)\n\n• Financial plan\n\n2. Once the scientific strength of the application has been determined then the strength of capacity strengthening should be assessed considering the following points:\n\n• Host institutions have capability and resolve to utilise the programme for the long-term development of their own research strategy/aspiration\n\n• Wider training programme for other researchers and laboratory technicians\n\n• Clearly research active lead members\n\n• Likelihood that support will lead to institutional changes and long-term benefits for the African partners\n\n3. Additional points to consider:\n\n• Consortium lead members who are female\n\n• Africa lead members that are early to mid-career scientists\n\n• Multi-disciplinary applications\n\n• Inclusion of non-Anglophone countries\n\nFor the round one panel meeting, two separate matrices were used to capture data on a) the contents of each application and b) panel members’ contributions and interactions (Supplementary file 1). The matrices ensured data were captured for assessment criteria areas of scientific excellence, research capacity strengthening and additional criteria (e.g. gender, career stage). It also enabled information to be captured on the selection process and panel members’ contributions (e.g. the process and format used to discuss each application, time spent on various aspects of each application including applicants’ demographic and professional backgrounds, details of how and which assessment criteria were used and discussed and other communication among the panel members).\n\nFor the round two panel meeting, the observation matrices were revised based on lessons learned from observation of the round one meeting. The main changes were to combine the two matrices into one with sub-divisions for key assessment themes (scientific strength, research capacity strengthening, other assessment criteria, themes not covered in assessment criteria). This matrix was completed for each shortlisted application as it was discussed by the panel members (Supplementary file 2). The observation of the round two panel meeting used the same researcher’s observation guide as in round one to ensure methodological consistency. As in round one, the round two observation focussed on processes and contents, verbal and non-verbal communication of panel members and any other relevant observations, with particular emphasis on which and how assessment criteria were used. This was because the award-making institution had made adjustments to the assessment criteria - to improve clarity and to make it compulsory to include strengthening of laboratories - based on our recommendations after the first panel meeting.\n\nData management and analysis of panel meeting observations. All the researchers’ hand-written observation notes were transferred onto an Excel spreadsheet. These data were coded and analysed under the a priori categories of ‘assessment criteria’ and ‘other observations’. Items with the same code that emerged from the data were amalgamated into themes during debriefing discussions among the researchers after each panel meeting observation. This process facilitated a balanced interpretation of the data and helped reduce the subjectivity of the researchers’ interpretations. The frequency with which key items were discussed at the meeting was also calculated. The datasets concerning the observations of the panel meetings have not been made available because they contain sensitive information and it was not possible to anonymise them without losing the meaning of the data.\n\nSemi-structured interviews (Supplementary file 3) with nine (six in round one, three in round two) purposively selected panel members were conducted after each of the two meetings to explore their views and perceptions of the award-making process. The choice of panel members to interview was designed to maximise diversity in nationality, expertise profile and gender. Interviews were conducted by two researchers by phone/Skype within three months of the panel meeting.\n\nThe assessment criteria were also used to inform the guides used to interview panel members. The interview guide topics included panel members’ experiences and perceptions of the award selection process, their role in the panel and their understanding of scientific excellence, research capacity strengthening and research partnerships. Since these interviews revealed some differences between interviewees’ responses, the interview guide was revised after round one so that these differences could be explored in more depth during the round two interviews. Interviews lasted 30–60 minutes. They were audio-recorded (with permission) and the interviewers took notes summarising the main issues discussed under each topic (Dataset 1). Interviewees were assured that the information they gave would be treated confidentially and anonymously to avoid the possibility that panel members’ quotes could be attributable. Since the number of interviewees was small, demographic details for interviewees’ quotes have not been provided in order to maintain anonymity.\n\nData management and analysis of interviews. All hand-written notes were transcribed electronically and checked against the recordings for accuracy. The data in the notes were then coded using codes which were developed iteratively based on themes that emerged from the transcripts and agreed among the research team. Once coding had been completed, links within and between codes were explored and data were grouped into higher level themes to allow for data interpretation and explanation. The panel guidance notes were also used in the analysis to determine how they were understood and followed by panel members during the meetings.\n\nThe study was approved by the Liverpool School of Tropical Medicine Research Ethics Committee (ref Research Protocol 13.14RS)\n\n\nFindings\n\nIn line with the developmental nature of this study, the findings are presented as a narrative, following the chronological order of the events covering the pre-award (document review), award-making (observation of panel meetings) and post-award (interviews with panel members) stages.\n\nPre-award process. The award-making institution received 26 eligible applications for round one of the programme grant. During the initial assessment stage, each panel member was allocated a selection of the eligible applications based on their area of expertise. Each application was assessed independently by three panel members using scores between one (poor) and seven (outstanding) against the assessment criteria. Fifteen applications were shortlisted for external review, six in water and sanitation, six in renewable energy and three in soil-related science. At least five external specialists assessed each application and their reports were sent to the panel members prior to the panel meeting. Their scores were collated and applications were ranked according to their average score. Each application, with its external specialists’ reports, was allocated to three of the panel members to lead on the discussion during the award-making meeting.\n\nAward-making panel meeting. The round one panel meeting started with a general introduction of all participants, followed by a presentation by the award making institution giving relevant background information about the programme and a brief outline of the selection criteria to be used when discussing the applications during the meeting (box 1).\n\nThese assessment criteria reflected those in the panel guidance notes. However, an indication of the differential weighting between ‘primary’ and ‘additional considerations’ was provided in the introductory presentation at the panel meeting but was not included in the written guidance the panel members received in advance of the meeting.\n\nProcess: The overall format of discussion of each application was consistent throughout the meeting. One panel member led on each proposal and presented their opinion of the application before the other two panel members contributed their comments. After the discussion of the application by the three panel members assigned to that application, all panel members were invited to express their views about the proposed project.\n\nApplications were assigned to each panel member according to their area of expertise while aiming to also provide diversity in terms of Francophone or Anglophone and UK or SSA-based. The observation revealed that the three panel members assigned to discuss a selected application demonstrated a thorough understanding of what was written in the proposals and took into account the applicants’ expertise and backgrounds. Panel members consistently made use of the external reviewers’ comments to inform discussions about the applications. A summary report of the discussions was provided for successful applicants.\n\nIn total panel members spent 189 minutes (excluding breaks and general discussions) discussing the 15 applications (average 13 minutes per application), spending an average of 15 minutes on applications they recommended for a grant and 11 minutes on proposals that were not successful. In one instance, panel members spent 20 minutes on a ‘maybe’ application that straddled the boundary between success and rejection. It was decided that ‘maybe’ applications would be discussed further after the panel members had gone through all proposals. The rationale was that this allowed for more informed decisions of ‘maybe’ applications as panel members could compare these to all other applications.\n\nContent: The observations revealed substantial variations in how each application was presented and therefore how it was discussed by the panel members. Panel members were not asked to use a specific format for presenting applications and, although most spoke to several of the assessment criteria, not all criteria were discussed for each application (see Table 1 for frequency of themes discussed for each round). Scientific excellence and innovation were given the greatest attention by panel members as well as the applicants’ credentials (e.g. number of publications, journal impact factors, frequency of publication) and success in obtaining grants, including prior collaborative projects.\n\nResearch capacity strengthening was discussed for almost all applications but for a shorter time and in less detail than scientific excellence. It was often used as an additional selection criterion to differentiate between two applications of similar quality. The observation revealed that panel members commonly used the term ‘capacity strengthening’ in relation to research training for individuals, including PhD candidates. There were limited discussions surrounding broader aspects of capacity strengthening such as PhD supervision plans and the institutional research infrastructure. Other aspects of some, but not all, applications were discussed by the panel, such as presentation of the application, budgets and applicant’s career age (i.e. research-active years since their PhD) especially in relation to their potential (in)experience of managing large awards.\n\nInterviews with panel members after the selection meeting showed that they were positive about the process and emphasised the high levels of rigour, collaboration and balanced expertise of the panel members. Their assessment was primarily based on their previous experiences of serving on other review panels. Responses to questions about how the panel worked as a team showed that all those interviewed agreed that the committee was cooperative and respectful and that panel members were given enough time to speak and listened to each other. Interviewees highlighted that for each application consensus was reached based on thorough and constructive discussions. A few interviewees attributed the success of the panel to effective chairing of the meeting.\n\nSpecific expertise in one or more of the three priority research areas of the programme was quoted as the main reason why panel members believed they were approached to participate in the review process. One panel member also thought she had been selected because she was female and an African panel member felt he was invited because he could provide an African perspective and contextual expertise. He noted that during the review process he ‘scrutinised the applications with regard to development relevance for Africa’.\n\nMost interviewees felt that they were provided with clear guidelines about how to review the applications. However, interview data mirrored the researchers’ observations that for some aspects panel members had different understandings about the interpretation or weighting of the assessment criteria, particularly relating to the balance between scientific excellence and research capacity strengthening, as the following two quotes exemplify:\n\n‘… the starting point was quality of science … the tiebreaker was actually the capacity building aspect. Maybe naïvely I thought it would be the other way round … So I was perhaps surprised that the quality of science dominated …’ (Interviewee 5)\n\n‘It could have been the best science ever, but if it did not have the capacity building, it wasn’t going to get funded. I did feel genuinely that we were steered to look at that and give that significant weighting.’ (Interviewee 3)\n\nInterview data showed that panel members had a common understanding of scientific excellence, focussing primarily on innovative research projects and the applicants’ publication and grant records. However, responses to interview questions about what they thought were key characteristics of research capacity strengthening showed a range of different responses. Characteristics mentioned by the various panel members included: sustainable physical and human infrastructure of institutions, multi-disciplinary and international collaborations, training of researchers at different levels (including students, postdoctoral researchers and supervisors), research uptake strategies and sustainable monitoring and evaluation processes. There did not appear to be a common understanding of the concept of ‘research capacity strengthening’ among the panel members.\n\nLearning generated by developmental evaluation of round one selection process and used to influence round two. Recommendations for improvements to the selection panel process for round two were derived from the findings from research into round one selection processes (box 2). These recommendations included improving the clarity and specificity of assessment criteria guidelines, a more standardised structure for presenting each application and increasing the diversity of the panel.\n\nPre-award process. The shortlisting process for the 47 eligible applications was the same as for round one except for the use of revised guidance on assessment criteria (see box 3).\n\nAward-making panel meeting. The overall format of the panel meeting was similar to round one with presentation of the applications by the relevant lead panel members, followed by open discussion by the whole panel.\n\nProcess. Compared to round one, there was less variation in how the applications were discussed. In total panel members spent 178 minutes (excluding breaks and general discussions) discussing the shortlisted 19 applications (average 9 minutes/application versus 13 minutes/application in round one). The relative time spent on successful, unsuccessful and ‘maybe’ applications was similar to that in round one. Panel members spent on average 11 minutes on the 5 applications they recommended for a grant (15 minutes in round one), 9 minutes on the 14 unsuccessful proposals (11 minutes in round one) and 14 minutes on the ‘maybe’ applications (20 minutes in round one).\n\nContent. Compared to round one there was less variation in the focal areas of the discussions concerning scientific strength. These areas covered reviewers’ scoring, the novelty of the research, research methods and how well individual research projects were integrated within a consortium (see Table 1 for frequency of themes discussed for each round). The panel also discussed areas not covered in round one and not included in the revised assessment guidance, such as dissemination of research findings, transferability of results and risk assessments. Compared to round one there was also more discussion on development relevance for Africa and greater consensus among the panel regarding applications recommended and not recommended for funding.\n\nThere were no major differences between round one and round two in how panel members discussed the credentials of scientists and their complementarity within consortia. Discussions focused on applicants’ complementary research backgrounds, especially their publication and grant records, frequently using the term ‘well-published’.\n\nResearch capacity strengthening components of applications were discussed in 13 out of the 19 applications; these were not necessarily those in which scientific strength was considered to be possibly insufficient. The panel considered many of the points under research capacity strengthening that were listed in the revised guidance notes (e.g. relevance of training, equipment and statistics training, cross partner training). However, the training of laboratory technicians was only discussed once and there was limited consideration of how proposals would impact on the wider research infrastructure.\n\nAll three interviewees assessed the award-making process positively, commenting particularly on how well the panel worked as a group, the chairing of the meeting and how the panel made informed decisions about funding. They highlighted a high level of mutual respect among the panel members and an increased level of familiarity with the process based on experiences from round one. Despite using the revised assessment guidance notes in round two, the interviews revealed that there was still some ambiguity in the interpretation of the weighting of the assessment criteria. Whereas two panel members highlighted the importance of scientific strength, noting ‘we have to select science, capacity strengthening was always the second tier’, another panel member (Interviewee 7) explained:\n\n‘When I review them [applications], I am likely to look at capacity enhancement first, rather than the science, and there is a little bit too much focus on science, and not on the capacity aspect or training aspect, the training programme.’\n\nOne panel member felt that capacity strengthening was given less attention during this panel meeting compared to the previous one, but in contrast another interviewee (Interviewee 8) noted:\n\n‘I think that [discussion of capacity strengthening] is one thing that I would say was stronger, the 2015 round compared to the 2014…..I think the panel really did appreciate and did really grasp some of the key issues in capacity strengthening this time to a significantly greater extent than during the previous occasion.’\n\nIn line with the panel guidance notes, interviewees noted that additional points, such as gender and the career level (i.e. early- or mid-career) of African scientists and non-Anglophone applications, were discussed in less detail than scientific strength and capacity strengthening.\n\n\nDiscussion\n\nThis study primarily used structured, overt observations and semi-structured interviews to explore the peer review process used by an award-making institution to select ten UK-Africa natural science research consortia over two annual rounds. The selection panel had been constituted to include expertise across the three different scientific areas (i.e. renewable energy, soil-related sceinces and water and sanitation), women and Francophone and African members. The panel constitution was generally consistent for both rounds of awards which facilitated inter-round comparisions and helped ensure consistency in standards between the two rounds. Using the same panel for both rounds had advantages in that during the second round they achieved consensus more quickly and increased their focus on development aspects in addition to scientific aspects. Consideration was given to increasing the diversity of the panel for round two, but this proved difficult given the relative paucity of women and African scientists in these three research areas. It was this capacity problem that the programme was designed to address, so it may be possible in future to expand the panel diversity. The peer review process is known to be influenced by the constitution of the panel with more deference between members of multi-disciplinary panels in which their expertise does not substantially overlap (Lamont & Huutoniemi, 2012), a finding which was confirmed by this study.\n\nThe shortlisting process was similar for both rounds of awards and broadly followed the benchmark process used by many grant-awarding bodies (RCUK, 2006). The benchmark describes how submitted proposals should be assessed by some combination of external referees, peer review panels and expert programme managers and final reports should be provided for successful applications. For this UK-Africa award, the final assessment panel comprised only scientists. Each eligible application was scored by three panel members. Those with the highest scores that were considered to have met the assessment criteria were shortlisted. Each shortlisted application was reviewed by at least five external reviewers and their reviews were considered alongside the panel members’ reviews in the final selection meeting. Scores for each application were pooled across panel members and then each application was discussed individually. There is evidence that pooling scores across a panel increases reliability compared to using scores produced by individuals reviewers (Fogelholm et al., 2012; Jayasinghe et al., 2006).\n\nThe study showed that panel members demonstrated a good understanding of the contents of the applications and took the relevance of applicants’ credentials into consideration in their decision making. Panel members themselves were positive about the process, particularly the rigour, collaboration and diversity of expertise. Compared to their experience on other review panels, some members noted that the task for this panel was particularly challenging because of the need to cover three different natural science areas and many different countries. Compared to round one, in round two there was more discussion on development relevance for Africa, suggesting increased awareness of its importance in meeting the goals of the programme. There was also greater consensus among the panel regarding applications to be recommended or not recommended for an award. The relative time spent on different types of applications was consistent between the two rounds. The shortest time was spent on unsuccessful applications and the longest time on those initially classified as ‘maybe’. This timing pattern has been observed elsewhere (Ahlquist et al., 2013) and suggests that negotiations about funding decisions are most critical for applications that are close to the boundary.\n\nIn general, the panel based their assessments of the applications on the guidance notes. However, even for round two there were some inconsistencies in the written and oral information provided to the panel members concerning differential weighting between primary and secondary considerations. In response to findings from the evaluation of the first round of award selection, the clarity and specificity of assessment criteria guidelines were improved. The subjectivity and lack of consistency in the use of criteria by reviewers can adversely affect the reliability and validity of the assessment. Since this may result in mistrust among grant applicants about the review process it has been found that it is important to ensure transparency in the review process by, for example providing definitions for each assessment criterion (Abdoul et al., 2012). A statistical model has been proposed to analyse peer review scores for grant applications which accounts for differences in reviewers scoring patterns, ratings from preliminary scores and group discussions and the final results. Initial findings from application of the model to a US peer review system suggested that it would have resulted in a 25% change in the funded proposals (Johnson, 2008). It is also recognised that the cognitive and professional perspectives of reviewers influence their decisions, so peer review cannot be an objective process characterised by a consistent application of a set of criteria (Lamont & Huutoniemi, 2012). The need for consistent criteria seems to be of more concern to science disciplines than social science and humanities, perhaps because the latter disciplines are more conscious of the effect of intersubjectivity (Lamont & Huutoniemi, 2012).\n\nPanel members were not given a specific format for presenting their summary reviews of applications. This resulted in significant variations in the sequencing and coverage of topics discussed for each application. Although scientific excellence and the applicants’ credentials were always discussed, some assessment criteria were not used at all for some applications. This may have been appropriate since, if the main criteria of high quality science and suitable applicants were fulfilled, then other factors may not have warranted consideration. However, it did mean that for a few applications, secondary criteria such as research capacity strengthening, gender and career stage, were not discussed. If the award-making institution wants such factors to be taken into account for every application in an unbiased non-subjective way, then it may be necessary to introduce a scoring system, with clear definitions for each score, as part of the panel meeting process.\n\nThere were significant variations in the panel members’ understanding of the term ‘research capacity strengthening’, which mirrors the current lack of clarity regarding the definition of this term in the literature (Gadsby, 2011). When the UK-Africa programme was first conceived, the capacity strengthening element focused on training for individuals. However since then the importance of the research environment in enabling researchers to utilise their training effectively has been increasingly recognised. This has resulted in a shift in research funders’ focus towards strengthening institutions’ research systems and infrastructure (Bates et al., 2014; ESSENCE, 2014). To facilitate a common understanding of the concept of ‘research capacity strengthening’ the panel would have benefitted from clearer guidance about the definition as applied to this programme and more detailed assessment criteria. In evaluating complex applications that address the dual goals of high quality science and research capacity strengthening there is a tension between having enough assessment criteria to be able to evaluate contextual and collaborative variables without over-burdening the reviewers and making the assessment process too cumbersome. For panels that have to assess these dual goals there is also a balance to be struck between traditional scientific criteria and criteria related to, for example, research culture and infrastructure, that are important to achieve training and capacity building objectives (Wooten et al., 2014).\n\nThe overt observation method was chosen because it would yield a rich amount of data when exploring the award-making process, how panel members used the guidance notes and how the two rounds of award-making differed from each other. The presence of more than one researcher taking independent notes at each meeting and discussing findings among researchers immediately after the meeting promoted an unbiased interpretation of the data and enabled the researchers to document processes and interactions that the panel members themselves might consider self-evident or not worth-mentioning. However, overt observation does not allow exploration of the cause of observed phenomena and it is not possible to be confident that observations represent normal behaviours since subjects may act differently when they know they are being watched (McNaughton et al., 2013). To mitigate this possibility the observation data were compared with information from the semi-structured interviews with panel members. The interviews provided information about experiences of the panel members which could not be captured through observations. It is possible that interviewees might have given responses that did not truly reflect their feelings as they were aware that information might be shared (anonymously) with the award making institution. Therefore, before each interview the interviewees were reassured that their information would be treated confidentially and anonymously.\n\n1. The constitution of the panel should be diverse enough to reflect the scientific topics, LMIC context, gender and language of the applications and should be maintained across different rounds of awards to ensure consistency.\n\n2. The shortlisting and selection process, with involvement of external reviewers, pooled scores and discussions during the panel meeting, is in line with funders’ benchmarks and can be applied to these complex consortia applications to provide a rigorous and equitable selection mechanism.\n\n3. Assessment criteria need to be defined in terms of content and their weighting. The number of assessment criteria should be limited to those most revelvant to the programme’s aims to enhance consistency during the discussions.\n\n4. The guidelines should specify whether criteria such as gender and career stage must be considered in all cases, or whether they should only be used as ‘tie breakers’ and how much weight they should carry.\n\n5. For programmes which have dual (but not exclusive) aims of generating high quality research and strengthening research capacity:\n\na. the relative weighting of each of the goals needs to be made explicit\n\nb. assessment guidelines need to be clear about how to evaluate the research capacity strengthening component of applications and panel members need to all have the same understanding of what research capacity strengthening means in the context of the programme.\n\n\nConclusions\n\nThis study provides an evaluation of the complex process of awarding grants to multi-national, multi-disciplinary science research consortia. It provides insights into how the award process was designed and conducted and gauged how closely the process matched the overall aims of the programme which were to support both high quality science research and development of research capacity in the African institutions. Research consortia are one of the most popular ways of funding collaborative multi-national research because, compared to a single site study, they provide additional benefits such as greater generalisability of findings and a more comprehensive understanding of the issues. Multi-disciplinary consortia can also create synergies that make them more influential in catalysing changes in policies and programmes and they can help to address inequality of resources and research opportunities among partners. The diversity and complexity of multi-partner consortia presents challenges and potential risks, such as inequity between partners and lack of cohesion around common goals and expectations (Dean et al., 2015), that need to be considered in the selection process. It is important for award-making institutions involved in these complex, consortia-based research models to put in place mechanisms for robust and systematic learning and to be flexible enough to incorporate changes into subsequent selection processes to make the award-making process more effective.\n\n\nData availability\n\nDataset 1: Notes of observations from round 1 and round 2 panel meetings DOI: 10.5256/f1000research.12496.d178727 (Gregorius et al., 2017)\n\nTranscriptions of interviews with panel members are available from the corresponding author on request. They have not been made available as a dataset because they cannot be de-identified without compromising anonymity and the ethical approval conditions for the project stated that only the research team would have access to the data.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded through the Royal Society by the Department for International Development, UK (grant number 203041-101). The views expressed in this publication are those of the authors and not necessarily those of the Royal Society or the Department for International Development.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe are very grateful to Dr Helen Smith of the Liverpool School of Tropical Medicine for her valuable contributions to the early stages of this study.\n\n\nSupplementary material\n\nSupplementary file 1: Observation matrixes Round 1\n\nClick here to access the data.\n\nSupplementary file 2: Observation matrixes Round 2\n\nClick here to access the data.\n\nSupplementary file 3: Interview guides for panel members for round 1 and round 2 awards\n\nClick here to access the data.\n\n\nReferences\n\nAbdoul H, Perrey C, Amiel P, et al.: Peer review of grant applications: criteria used and qualitative study of reviewer practices. PLoS One. 2012; 7(9): e46054. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "26762", "date": "03 Nov 2017", "name": "Yaso Kunaratnam", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a timely, well-written and important look at the under-researched area of evaluating peer review for multi-national consortia.\n\nThe rise in spend on global development research has meant that complex consortia models are increasingly being used, and the findings will be useful for funders to think about how they could improve effectiveness and practice in this area. A synthesis on the key findings and lessons learned (what works well e.g. pooling scores/what does not work well) would be really useful.\n\nSetting up dual-purpose consortia to achieve scientific excellence and strengthen research capacity is a popular model, and I am not surprised to see tensions and conflicting views on which are more important. As the authors suggest, weighting would help assessment. I also think that research capacity experts could be on future peer review panels to address some of the gaps mentioned in levels of understanding on the topic.\n\nThe article highlights that multi-national dual-purpose consortia is a complex model to assess – it was interesting to see the number of areas that peer reviewers take into consideration in table 1. I could see a useful resource emerging to help funders prioritise. I agree that assessment criteria should prioritise areas most closely linked with the goals of the programme – it is a challenge however when there are so many considerations of interest to funders e.g. interdisciplinarity, equitable partnerships, ethics, research uptake/impact, sustainability of networks, expertise within the consortia to deliver on the dual-purpose.\n\nThe authors could suggest further research or work that is needed to improve peer review. It would be good to know how peer reviewers are sourced to help inform how to address gaps in the diversity of panels, and also look at the quality of assessment guidelines and how they could be improved, e.g. could they offer frameworks to assess quality that are evidence-based. Beyond peer review, a broader look at whether dual-purpose consortia are the right model for purpose or how they could be improved would be valuable.\n\nThe methodology is sound, with and limitations explained and ethical considerations approved. Recent and relevant literature is referenced.\n\nMinor comments include:\nIntro para 4 - sustainable research networks mentioned as a goal but there is no reference later in terms of assessment.\n\nTable 1 could include overall total for scientific strengths, capacity and other areas.\n\n1st para under Discussion – last sentence on Lamont & Huutoniemi, 2012, I did not quite understand.\n\nLinks to the Swedish Research Council publication and statistical model would be useful, and funder assessment guidelines if they can be made accessible.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3285", "date": "22 Dec 2017", "name": "imelda bates", "role": "Author Response", "response": "Response to Yaso Kunaratnam, UK Collaborative on Development Sciences (UKCDS), London, UK This is a timely, well-written and important look at the under-researched area of evaluating peer review for multi-national consortia. The rise in spend on global development research has meant that complex consortia models are increasingly being used, and the findings will be useful for funders to think about how they could improve effectiveness and practice in this area. A synthesis on the key findings and lessons learned (what works well e.g. pooling scores/what does not work well) would be really useful. Setting up dual-purpose consortia to achieve scientific excellence and strengthen research capacity is a popular model, and I am not surprised to see tensions and conflicting views on which are more important. As the authors suggest, weighting would help assessment. I also think that research capacity experts could be on future peer review panels to address some of the gaps mentioned in levels of understanding on the topic.Response: We have now mentioned the need to include research capacity experts on future peer review panels The article highlights that multi-national dual-purpose consortia is a complex model to assess – it was interesting to see the number of areas that peer reviewers take into consideration in table 1. I could see a useful resource emerging to help funders prioritise. I agree that assessment criteria should prioritise areas most closely linked with the goals of the programme – it is a challenge however when there are so many considerations of interest to funders e.g. interdisciplinarity, equitable partnerships, ethics, research uptake/impact, sustainability of networks, expertise within the consortia to deliver on the dual-purpose.Response: We have indicated that, if confirmed by further studies, the approach used in table 1 might be useful to help funders prioritise The authors could suggest further research or work that is needed to improve peer review. It would be good to know how peer reviewers are sourced to help inform how to address gaps in the diversity of panels, and also look at the quality of assessment guidelines and how they could be improved, e.g. could they offer frameworks to assess quality that are evidence-based. Beyond peer review, a broader look at whether dual-purpose consortia are the right model for purpose or how they could be improved would be valuable.Response: It is clear that further work is needed to understand more about the peer review process such as how it compares to other models, and whether consortia are the right models. Although a detailed discussion about these topics is beyond the scope of this paper we have included a couple of references that deal specifically with evidence and research on this topic including a very recent systematic review (Guthrie, et al 2017; Barnett, et al 2015) The methodology is sound, with and limitations explained and ethical considerations approved. Recent and relevant literature is referenced. Minor comments include: Intro para 4 - sustainable research networks mentioned as a goal but there is no reference later in terms of assessment. Response: Networks were not discussed by the panel members and we have stated this in the revised version. Table 1 could include overall total for scientific strengths, capacity and other areas. Response: Table 1 has been amended 1st para under Discussion – last sentence on Lamont & Huutoniemi, 2012, I did not quite understand. Response: This sentence has been adjusted Links to the Swedish Research Council publication and statistical model would be useful, and funder assessment guidelines if they can be made accessible Response: Details of the assessment process including sections on criteria, openness and gender equality, are available on the Swedish research Council’s website at https://www.vr.se/inenglish/researchfunding/assessment/assessmentcriteria.4.7257118313b2995b0f27ace.html (accessed 15 December 2017)" } ] }, { "id": "28056", "date": "20 Nov 2017", "name": "Adrian Barnett", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt’s good to read a thoughtful reflection on how peer review panels function given their importance for research. This study looked at a funding scheme that covered a range of fields and had an important aim of funding international groups to work on overseas aid projects.\n\nIt would be worthwhile citing the recent systematic review in this area (Guthrie et al., 20171), which includes some additional references that consider the question whether panel meetings are worth the additional costs compared with independent reviews.\n\nThe panel’s gender split was nowhere near 50:50. This was commented on in the results in that it: “proved difficult given the relative paucity of women and African scientists in these three research areas.” Part of the criteria for funding is “Support for female scientists”, but the group organizing the panel is ironically not adhering to this. Taking part in a panel can benefit researchers as it is a tremendous way to learn how to write a good proposal. As Guthrie et al state, “Members of funding panels may also benefit directly from their membership.” Hence not selecting women on the panel may just perpetuate the “paucity” of female scientists in this area and the funding agencies could be a key group for addressing this problem.\n\nWas there any discussion of the funding line and how many proposals might be funded? This often shapes the discussion as if only a handful of proposals can be funded then the panels members know that only the very best will get funding.\n\nThe authors state: “There is evidence that pooling scores across a panel increases reliability compared to using scores produced by individuals’ reviewers (Fogelholm et al., 2012)” But the main conclusion of Fogelholm’s paper was: “panel discussions per se did not improve the reliability of the evaluation.”\n\nWhat order were proposals discussed in? The order of the day can matter (e.g., reviewers being more alert in the morning), and ideally the proposals should be assessed in a random order to remove any long-term biases (e.g., from alphabetical ordering).\n\nWhat feedback was given to applicants? This is an important part of the process and given that panels cost time and money to assemble it would be worth giving detailed feedback so that applicants can improve. A funding scheme that I was involved in gave audio transcripts of the panel meeting to applicants (Barnett et al., 2015) 2 , which was greatly appreciated by applicants.\n\nThis point maybe outside the scope of the paper, but there is a great emphasis on “excellence” in this funding scheme, but “excellence” has little meaning in research as pointed out here: https://www.nature.com/articles/palcomms2016105 3, and the authors of that paper conclude that “soundness and capacity-building” are far better criteria.\n\nMinor comments:\nDid any of the panel members also have applications being assessed?\n\nIt would be useful to include a link to the funding scheme or a list of the winning proposals.\n\nI was surprised to read the Journal Impact Factors were used, given that they are a poor proxy the individual quality of papers.\n\nThe authors state: “The peer review process is known to be influenced by the constitution of the panel with more deference between members of multi-disciplinary panels in which their expertise does not substantially overlap (Lamont & Huutoniemi, 2012), a finding which was confirmed by this study.” I did not read this as a major finding of this study and it was not highlighted in the results, nor was there any formal analysis that covered this. I think this needs expanding in the results to make such a strong statement in the conclusion.\n\n“There was also greater consensus among the panel regarding applications to be recommended or not recommended for an award” This finding was not shown in results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3284", "date": "22 Dec 2017", "name": "imelda bates", "role": "Author Response", "response": "Response to Adrian Barnett, Institute of Health and Biomedical Innovation, Queensland University of Technology, Kelvin Grove, Qld, Australia It’s good to read a thoughtful reflection on how peer review panels function given their importance for research. This study looked at a funding scheme that covered a range of fields and had an important aim of funding international groups to work on overseas aid projects. It would be worthwhile citing the recent systematic review in this area (Guthrie et al., 20171), which includes some additional references that consider the question whether panel meetings are worth the additional costs compared with independent reviews. Response: We agreed that this is a very relevant review and we have included some of its findings in the introduction and discussion sections. We too were surprised by the lack of evidence about the effectiveness of peer-review for guiding funding decisions and also the lack of thoughtful critique about the pros and cons of the process compared to other ways of making funding decisions. Hopefully the Guthrie et al paper may start to stimulate a discussion, and then action, about more cost-effective ways to reach funding decisions. The panel’s gender split was nowhere near 50:50. This was commented on in the results in that it: “proved difficult given the relative paucity of women and African scientists in these three research areas.” Part of the criteria for funding is “Support for female scientists”, but the group organizing the panel is ironically not adhering to this. Taking part in a panel can benefit researchers as it is a tremendous way to learn how to write a good proposal. As Guthrie et al state, “Members of funding panels may also benefit directly from their membership.” Hence not selecting women on the panel may just perpetuate the “paucity” of female scientists in this area and the funding agencies could be a key group for addressing this problem. Response: We have expanded discussion of the gender make-up of the panel in the discussion, and the potential consequences for lack of diversity of perpetuating inequality, and we have also mentioned of the role of funders in helping to make this happen. Was there any discussion of the funding line and how many proposals might be funded? This often shapes the discussion as if only a handful of proposals can be funded then the panels members know that only the very best will get funding. Response: The scheme was specifically designed and budgeted to fund 10 consortia – we have made this clear in the introduction text. The authors state: “There is evidence that pooling scores across a panel increases reliability compared to using scores produced by individuals’ reviewers (Fogelholm et al., 2012)” But the main conclusion of Fogelholm’s paper was: “panel discussions per se did not improve the reliability of the evaluation.” Response: We have altered the wording to more closely reflect the conclusion of this paper What order were proposals discussed in? The order of the day can matter (e.g., reviewers being more alert in the morning), and ideally the proposals should be assessed in a random order to remove any long-term biases (e.g., from alphabetical ordering). Response: This is an important consideration which forgot to mention in the paper – the applications were not discussed in any particular order (e.g. alphabetically) and we have included a statement to explain this What feedback was given to applicants? This is an important part of the process and given that panels cost time and money to assemble it would be worth giving detailed feedback so that applicants can improve. A funding scheme that I was involved in gave audio transcripts of the panel meeting to applicants (Barnett et al., 2015) 2 , which was greatly appreciated by applicants. Response: This sounds like a great idea and would potentially be very helpful to applicants so we have stressed the need to improve the quality of feedback to applicants and mentioned audio transcripts in the discussion. This point maybe outside the scope of the paper, but there is a great emphasis on “excellence” in this funding scheme, but “excellence” has little meaning in research as pointed out here: https://www.nature.com/articles/palcomms2016105 3, and the authors of that paper conclude that “soundness and capacity-building” are far better criteria. Response: We too are not comfortable with the idea of ‘excellence’ but this was the term used in the panel discussions. We have quoted the Nature reference you suggested in an attempt to encourage readers to think critically about this term in the context of research Minor comments: Did any of the panel members also have applications being assessed?Response: there were a couple of occasions when members declared a conflict of interest because they had a connection with the applicants. They were not present in the room during discussions about these applications It would be useful to include a link to the funding scheme or a list of the winning proposals. Response: In our ethics application for this research we gave an undertaking to maintain anonymity for the particiaptns (i.e. members) so we are not able to provide details of the scheme I was surprised to read the Journal Impact Factors were used, given that they are a poor proxy the individual quality of papers. Response: We agree that impact factors are increasingly recognised as a poor proxy for quality but we have presented the observations as we noted them at the time. The authors state: “The peer review process is known to be influenced by the constitution of the panel with more deference between members of multi-disciplinary panels in which their expertise does not substantially overlap (Lamont & Huutoniemi, 2012), a finding which was confirmed by this study.” I did not read this as a major finding of this study and it was not highlighted in the results, nor was there any formal analysis that covered this. I think this needs expanding in the results to make such a strong statement in the conclusion. Response: The original statement we included was inferred from some of the comments made by panel members during the interviews. However we recognise that an investigation of how multi-disciplinarity affects the review process was not an objective of this study and that the evidence to support this statement is not strong, so on reflection we have decided to remove it from the revised version. “There was also greater consensus among the panel regarding applications to be recommended or not recommended for an award” This finding was not shown in results. Response: We have expanded a couple of the sections to highlight findings that indicate greater consensus among panel members in round two. The relevant findings relate to a) less time needed to discuss each application and b) better consensus on a capacity strengthening focus. Is the work clearly and accurately presented and does it cite the current literature?Partly Is the study design appropriate and is the work technically sound?Yes Are sufficient details of methods and analysis provided to allow replication by others?Yes If applicable, is the statistical analysis and its interpretation appropriate? Not applicable Are all the source data underlying the results available to ensure full reproducibility?Partly Are the conclusions drawn adequately supported by the results?No References Guthrie S, Ghiga I, Wooding S: What do we know about grant peer review in the health sciences?. F1000Research. 2017; 6. Publisher Full Text 2. Barnett AG, Herbert DL, Campbell M, Daly N, Roberts JA, Mudge A, Graves N: Streamlined research funding using short proposals and accelerated peer review: an observational study.BMC Health Serv Res. 2015; 15: 55 PubMed Abstract \| Publisher Full Text 3. Moore S, Neylon C, Paul Eve M, Paul O’Donnell D, Pattinson D: “Excellence R Us”: university research and the fetishisation of excellence. Palgrave Communications. 2017; 3. Publisher Full Text" } ] } ]	1	https://f1000research.com/articles/6-1808
https://f1000research.com/articles/7-59/v1	16 Jan 18	{ "type": "Research Article", "title": "A comparison of physical activity and nutrition in young women with and without primary dysmenorrhea", "authors": [ "Dina Abadi Bavil", "Mahrokh Dolatian", "Zohreh Mahmoodi", "Alireza Akbarzadeh Baghban", "Dina Abadi Bavil", "Zohreh Mahmoodi", "Alireza Akbarzadeh Baghban" ], "abstract": "Background: Dysmenorrhea is the most common gynecological disorder in young women and is seen in almost 50% of women. The present study was conducted to investigate the relationship between physical activity and nutrition with primary dysmenorrhea in students at Mazandaran University of Medical Sciences (Sari, Iran) in 2015. Methods: This comparative descriptive study was conducted on 250 students with and without primary dysmenorrhea. Data were collected using personal-demographic, nutrition and physical activity questionnaires. The output was then analyzed in SPSS-18 using independent t-test, Chi-square test and logistic regression analysis. Results: The results showed significant differences between the two groups in terms of nutrition and physical activity, as the mean score of nutrition was 57.91 in the group with dysmenorrhea and 61.68 in the group without, while the mean intensity of physical activity was 5518.75 metric in the group with dysmenorrhea and 4666.42 metric in the group without. Physical activity was calculated by MET scale (minutes/week). This index measured the amount of consumed energy at the time of activity relative to that consumed at resting time. Conclusions: A healthier and more favorable nutrition style and more regular physical activity reduces the severity of dysmenorrhea in girls. Therefore, educational measures are required to raise awareness among young women about the effects of proper nutrition and physical activity on the prevention and reduction of dysmenorrhea complications.", "keywords": [ "Nutrition", "physical activity", "dysmenorrhea", "young women" ], "content": "Introduction\n\nPrimary dysmenorrhea is one of the most common gynecological disorders that refer to cramping pain in the lower abdomen during menstruation without pelvic pathology. This complication often occurs in the first and second years after the onset of menstruation during ovulation1. The overall prevalence of primary dysmenorrhea is 60% to 90% in adolescent girls but decreases with age2. Increased concentrations of prostaglandins and vasopressin, increased levels of leukotrienes and psychological factors are reported to be involved in the development of primary dysmenorrhea3. Prostaglandins cause pain by increasing uterine tone and contractions1. There are several medicinal and non-medicinal methods for improving or eliminating this complication. A non-medicinal treatment for primary dysmenorrhea is changing nutrition; for instance, reducing the intake of salt and animal fats, increasing the consumption of complex carbohydrates and dietary fibers and increasing physical activity4.\n\nAlthough various treatment methods have been proposed for this complication, there has been limited success. Some studies have proposed factors such as dietary habits5 nutrition6 and aerobic exercise7 as effective in the treatment of dysmenorrhea, but one study found no relationship between exercise and dysmenorrhea8. Since medicinal therapies can have side-effects, and as some people prefer to not be medicated, researchers and young women are both seeking alternative therapies for this condition9. The disparity of findings on this disorder led to the present study about nutrition and physical activity and their relationship to primary dysmenorrhea in university students, so as to facilitate interventions targeting nutrition and physical activity in young women.\n\n\nMethods\n\nThe present comparative descriptive study was conducted on 250 female students at Mazandaran University of Medical Sciences (Sari, Iran). Students were recruited during lectures at the university. Students with menarche who had menstrual pain and without pelvic pathological disorders and this pain limited to menstrual periods were classified as primary dysmenorrhea, which was self-reported.\n\nSampling lasted from late August to late November 2015. A total of 125 students belonged to the case group with primary dysmenorrhea and 125 students to the control group without this condition were case-matched to the study group through convenience sampling. The inclusion criteria for the cases consisted of being single, age 18 to 26, having moderate or severe (scores 4 to 10) and painless (scores 0 to 3) primary dysmenorrhea based on the McGill Pain Index, having no known chronic diseases, such as diabetes, hypertension, underlying cardiac diseases, infectious diseases, etc., having no self-reported symptoms such as burning, itching and abnormal vaginal discharge, and having no history of gynecological surgeries.\n\nSample size was calculated using the formula:\n\nData were collected using personal-demographic, nutrition and physical activity questionnaires (Supplementary File 1), the McGill Pain Index and height was measured by a metal ruler. The questionnaires were distributed by face to face interview. The personal-demographic questionnaire inquired about participants’ personal information, menstruation history, obstetric history and socio-economic status. The intervals of menstruation in a period of less than 21 days between 21 to 35 days or more than 35 days, according to the response of each person were marked. After obtaining the frequency, the mean of these were calculated in the two groups\n\nThe socio-economic status questionnaire contains 12 questions that were calculated using factor analysis method. Factor scores = 04754/0 * Education + 0/12080 * Assets + 0/34570 * Mother’s education + 0/27104 * Father’s education + 0/3585 * Type of home + 0/02277 * House size + 0/00403 * Number of residents At home - 0.06260 * Owning a private home 0/23442 * Mother’s income + 14.176 / 0 * Father’s income 0.04896 * Occupation. Using the above relationship, the socioeconomic score of each person was calculated.\n\nThe nutrition questionnaire consisted of 16 items that were scored based on a four-point Likert scale, with scores ranging from 16 to 64: never, 1; sometimes, 2; often, 3; always, 4. Questions 13 to 16 are never, 4; sometimes, 3; often, 2; always, 1. The nutrition questionnaire scores increased to a percentage and scores less than 33.3% of the total score of nutrition indicated poor nutrition, scores between 33.3% and 66.6% indicated somewhat proper nutrition and scores higher than 66.6% indicated good nutrition. Percentages were calculated as follows: Nutrition % = ((q1 + q2 + q3 + q4 + q5 + q6 + q7 + q8 + q9 + q10 + q11 + q12 + q13 + q14 + q15 + q16) - 16) / (64 - 16))* 100. The nutritional style questionnaire was used previously by Mahmoodi et al. for designing and psychometric evaluation. The Pearson correlation coefficient was 0.97. The Cronbach’s alpha coefficient in the nutrition aspect was 0.76, which confirmed its reliability and validity10.\n\nThe physical activity dimension of participants’ lifestyle was assessed using the long-form International Physical Activity Questionnaire [IPAQ; http://youthrex.com/wp-content/uploads/2017/06/IPAQ-TM.pdf]11, developed in 1998 by the WHO and CDD in Geneva as an international physical activity assessment tool for the age group 15 to 69. This version of the questionnaire consists of 27 items and reports physical activity levels in MET-minute/week and classifies people into three groups: A low activity group (less than 600 MET), a moderate activity group (between 600 and 3000 MET) and a high activity group (over 3000 MET) groups. The IPAQ is a global standard questionnaire whose validity and reliability have been approved in previous studies through content validity and Cronbach’s alpha12–15.\n\nThe McGill Pain Index is the most common visual analogue scale used in studies with an approved reliability and validity16.\n\nFor data collection, the researcher (DAB) visited the study settings and obtained the permission of the directors of the centers. She conducted preliminary interviews with the participants (briefed them on the study objectives and the confidentiality of the data before they submitted their informed written consents). Eligible candidates were then selected for participation in the study.\n\nData were analyzed in SPSS-18 using descriptive and analytical statistics such as mean and standard deviation, the independent t-test, the Chi-square test, Fisher’s Exact Test, Mann-Whitney’s U-test and the multiple logistic regression analysis.\n\nThe study was conducted after obtaining the approval of the Ethics Committee of Shahid Beheshti University of Medical Sciences (ID: SBMU2.REC.1394.102). The authors obtained the consent of Mazandaran University of Medical Sciences for doing this research. Written informed consent was obtained from all the participants.\n\n\nResults\n\nThe results showed significant differences between the two groups in terms of age (P=0.001) and degree of education (P=0.011), but not in terms of BMI (p=0.296), age at menarche (p=0.374), duration of menstrual cycles (p=0.540) and intervals between menstrual cycles (p=0.054), which means that the two groups matched in terms of these four variables (Table 1).\n\nIn the group with dysmenorrhea, the good nutritional status was 21.6% and in the non-affected group it was 36%. According to the scores obtained in the questionnaires, the two groups were significantly different in terms of nutrition score (p=0.008) and physical activity (p=0.011); (Table 2). The logistic regression analysis, however, showed no significant differences between the groups in terms of nutrition. The results showed a 1% reduction in the incidence of dysmenorrhea per each unit of increase in physical activity score; that is, a higher level of physical activity reduces the incidence of dysmenorrhea. Age also reduces the incidence of dysmenorrhea by 18%; in other words, the higher the age, the lower the incidence of dysmenorrhea (Table 3).\n\nNutrition score was calculated using a four-point Likert score, while physical activity was calculated by MET (minutes/week).\n\n\nDiscussion\n\nThe results showed that nutrition and physical activity were related to dysmenorrhea in the two groups. According to the results of Table 2, there was a significant difference between the two groups in terms of nutritional style (p = 0.008), physical activity (p = 0.11), but when some variables were adjusted by logistic regression analysis, nutrition didn’t show any difference between the two groups.\n\nAn optimal nutrition was found to reduce the severity of dysmenorrhea. In 1992, Ekstrom et al.17 showed that, during menstruation, hypertonic saline infusion increases vasopressin and oxytocin, and along with the increase in these two hormones, the severity of dysmenorrhea also increases. Increased prostaglandin was proposed as the main reason for the pain and excessive bleeding experienced1. Food items rich in magnesium can reduce the severity of dysmenorrhea by reducing the synthesis of prostaglandins and decreasing muscle and small vessel spasms18. Following a high-fiber diet can increase sex hormone-binding globulins and thus reduce the synthesis of prostaglandins, which are the main cause of dysmenorrhea19. Studies show that the arachidonic acid in animal fat is involved in the synthesis of prostaglandins, and therefore, foods such as meat and dairy are the main source of arachidonic acid5. Regarding the link between the daily use of the four food groups and dysmenorrhea, it can be argued that the high consumption of fish, eggs, vegetables and fruits is associated with a low incidence of painful menstruation20. Eliminating salty foods will decrease the incidence of dysmenorrhea as well21. Having breakfast every morning22 and eating nuts, pure honey24,25 are also effective in reducing the incidence of dysmenorrhea. The compound oleocanthal in extra virgin olive oil suppresses prostaglandin synthesis; in other words, it inhibits the enzymatic pathway for pain26.\n\nExercise acts as a non-specific analgesia by improving pelvic blood circulation and stimulating the release of beta-endorphins9. Exercise leads to the prevention and regression of dysmenorrhea by reducing stress and improving mood. Age at menarche is significantly higher in athletes27. Exercise reduces body fat, and since obesity is associated with a high prevalence of dysmenorrhea, the loss of fat significantly increases age at menarche27. Exercising three days before the beginning of the menstruation improves pelvic blood flow, disrupts the accumulation of prostaglandins in this part of the body and thus delays the onset of pain. Exercise during menstrual pain also leads to the faster transfer of excess substances and prostaglandin from the uterus, which is the main factor responsible for menstrual pain, and thus reduces the duration of pain during menstruation28. Exercise can reduce the activity of the sympathetic nervous system and increase the activity of the parasympathetic nerves during rest and reduce stress and thereby menstrual symptoms29. Regular aerobic exercise can reduce pain by increasing the secretion of endorphins, which are the most powerful natural opiates in the body30.\n\nSalehi et al. found a significant difference in the intensity and duration of pain after eight weeks of Pilates exercise between the intervention and control groups. On the first three days of menstruation, 30 minutes of brisk walking per day reduces primary dysmenorrhea pain. Dysmenorrhea was less prevalent in those who had regular exercise three sessions per week compared to those who did not exercise31. Exercise is most effective in the prevention of dysmenorrhea when it begins before the first menstruation and remains a fixed part of the adult’s lifestyle27. The present study showed that dysmenorrhea was less prevalent in those who were more physically active, and regular exercise can reduce stress in women and thus improve blood circulation and increase the amount of endorphins and neurotransmitters32. Educational and counseling measures are needed to emphasize the importance of exercise.\n\nThe two groups were significantly different in terms of age. The prevalence of primary dysmenorrhea decreased with age. This condition is prevalent between ages 20 and 24 and then progressively declines in prevalence after this age33. The two groups were not different in terms of BMI. Haidari et al. also showed no significant relationships between dysmenorrhea and the variables of BMI, height, weight and the waist-to-hip ratio34. A positive relationship has been observed between a high BMI and dysmenorrhea. The inconsistency between the results obtained by Harlow35 and those of the present study may be due to the fact that BMI is affected by factors such as race, age and gender and is therefore not a proper indicator of obesity, especially in athletes who have a high body mass31.\n\nIn this study, no significant relationships were observed between the two groups in terms of age at menarche, the duration of menstrual cycles and intervals between menstrual cycles. Nevertheless, Espiroff found a significant relationship between age at menarche and the intensity of primary dysmenorrhea2. The incidence of primary dysmenorrhea increases with longer intervals between menstrual cycles34, heavy menstrual bleeding33 and a menstruation lasting more than seven days36. Chung et al.37, however, argued that the duration of menstrual cycle is not related to dysmenorrhea. In the present study, the two groups were matched for confounding factors and there were therefore no differences between them in terms of menstruation history.\n\n\nConclusion\n\nDysmenorrhea is a cyclical and debilitating process. Due to its negative impact on quality of life, preventive and supportive measures are necessary in young women by raising awareness and promoting education about better lifestyles, which encompass proper nutrition and regular physical activity.\n\n\nData availability\n\nDataset 1: Raw data behind the results of this study. The coding schema for the data can be found in Supplementary File 2. DOI, 10.5256/f1000research.12462.d18927538", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThis research was derived from an M.S. thesis of Dina Abadi Bavil. We appreciate the cooperation of the honorable Research Deputies at the University of Shahid Beheshti and Sari University, as well as all students who participated.\n\n\nSupplementary material\n\nSupplementary File 1: Socio-demographic, nutrition and physical exercise questionnaires.\n\nClick here to access the data.\n\nSupplementary File 2: Coding schema for Dataset 1.\n\nClick here to access the data.\n\n\nReferences\n\nBerek JS, Novak E: Berek and Novak’s Gynecology. Wolters Kluwer Health/Lippincott Williams & Wilkins. 2012. Reference Source\n\nSperoff L, Fritz MA: Clinical gynecologic endocrinology and infertility. lippincott Williams & wilkins, 2011. Reference Source\n\nDeCherney A, Nathan L, Goodwin TM, et al.: Current Diagnosis and Treatment: Obstetrics and Gynecology 11e Inkling Chapter. McGraw Hill Professional. 2012. Reference Source\n\nO’Brien PS, Abukhalil IE: Randomized controlled trial of the management of premenstrual syndrome and premenstrual mastalgia using luteal phase-only danazol. Am J Obstet Gynecol. 1999; 180(1 Pt 1): 18–23. PubMed Abstract \| Publisher Full Text\n\nMolazem Z, Alhani F, Anooshe M, et al.: Epidemiology of dysmenorrhea with dietary habits and exercise. Zahedan Journal of Research in Medical Sciences. 2011; 13(3): 41–5. Reference Source\n\nKawabata A: Lipid Mediators and Pain Signaling. Biol Pharm Bull. 2011; 34(8): 1170–3.\n\nMohammadi B, Azamian Jazi A, Fathollahi Shourabeh F: The Effect of Aerobic Exercise Training and Detraining on Some of the Menstrual Disorders in Non-athlete Students in Lorestan Universities. The Horizon of Medical Sciences. 2012; 18(2): 5–12. Reference Source\n\nChung FF, Yao CC, Wan GH: The associations between menstrual function and life style/working conditions among nurses in Taiwan. J Occup Health. 2005; 47(2): 149–56. PubMed Abstract \| Publisher Full Text\n\nProctor M, Farquhar C: Diagnosis and management of dysmenorrhoea. BMJ. 2006; 332(7550): 1134–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMahmoodi Z, Karimlou M, Sajjadi H, et al.: Development of mother's lifestyle scale during pregnancy with an approach to social determinants of health. Glob J Health Sci. 2013; 5(3): 208–19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCommittee IR: Guidelines for data processing and analysis of the International Physical Activity Questionnaire (IPAQ). Consultado em. 2005; 20. Reference Source\n\nDinger MK, Oman RF, Taylor EL, et al.: Stability and convergent validity of the Physical Activity Scale for the Elderly (PASE). J Sports Med Phys Fitness. 2004; 44(2): 186–92. PubMed Abstract\n\nHazavehei SM, Asadi Z, Hassanzadeh A, et al.: Comparing the effect of two methods of presenting physical education Π course on the attitudes and practices of female Students towards regular physical activity in Isfahan University of Medical Sciences. Iran J Med Educ. 2008; 8(1): 121–31. Reference Source\n\nKurtze N, Rangul V, Hustvedt BE, et al.: Reliability and validity of self-reported physical activity in the Nord-Trøndelag Health Study: HUNT 1. Scand J Public Health. 2008; 36(1): 52–61. PubMed Abstract \| Publisher Full Text\n\nWoolven L: The Smart Woman’s Guide to PMS and Pain-Free Periods. John Wiley & Sons. 2010. Reference Source\n\nWall PD, Jones M: Defeating pain: The war against a silent epidemic. Springer. 2013. Reference Source\n\nEkström P, Åkerlund M, Forsling M, et al.: Stimulation of vasopressin release in women with primary dysmenorrhoea and after oral contraceptive treatment--effect on uterine contractility. Br J Obstet Gynaecol. 1992; 99(8): 680–4. PubMed Abstract \| Publisher Full Text\n\nHudson T: Natural progesterone: Clinical indications in women’s health. Female Patient. 2001; 26(4): 43.\n\nBarnard ND, Scialli AR, Hurlock D, et al.: Diet and Sex-Hormone Binding Globulin, Dysmenorrhea, and Premenstrual Symptoms. Obstet Gynecol. 2000; 95(2): 245–50. PubMed Abstract \| Publisher Full Text\n\nBalbi C, Musone R, Menditto A, et al.: Influence of menstrual factors and dietary habits on menstrual pain in adolescence age. Eur J Obstet Gynecol Reprod Biol. 2000; 91(2): 143–8. PubMed Abstract \| Publisher Full Text\n\nDurain D: Primary dysmenorrhea: assessment and management update. J Midwifery Womens Health. 2004; 49(6): 520–8. PubMed Abstract \| Publisher Full Text\n\nFjerbæk A, Knudsen UB: Endometriosis, dysmenorrhea and diet--What is the evidence? Eur J Obstet Gynecol Reprod Biol. 2007; 132(2): 140–7. PubMed Abstract \| Publisher Full Text\n\nCraig CL, Marshall AL, Sjöström M, et al.: International physical activity questionnaire: 12-country reliability and validity. Med Sci Sports Exerc. 2003; 35(8): 1381–95. PubMed Abstract \| Publisher Full Text\n\nWhite JW Jr: Detection of honey adulteration by carbohydrage analysis. J Assoc Off Anal Chem. 1980; 63(1): 11–8. PubMed Abstract\n\nBertelli D, Lolli M, Papotti G, et al.: Detection of honey adulteration by sugar syrups using one-dimensional and two-dimensional high-resolution nuclear magnetic resonance. J Agric Food Chem. 2010; 58(15): 8495–501. PubMed Abstract \| Publisher Full Text\n\nRezaeyan M, Abdali N, Araban M: Comparing analgesic effects of extra virgin olive oil and Ibuprofen on the intensity of primary dysmenorrhea: A crossover clinical trial. Iranian Journal of Nutrition Sciences & Food Technology. 2014; 9(2): 67–74. Reference Source\n\nSargolzayi M, Keykhayi N: dyssmenorhea and exercise in women. Journal of medical science. 1377; 3(11–12): 52–5.\n\nSalehi F, Marefati H, Mehrabian H, et al.: Effect of pilates exercise on primary dysmenorrhea. Journal of Research in Rehabilitation Sciences. 2012; 1(1): 248–53.\n\nGannon L: The potential role of exercise in the alleviation of menstrual disorders and menopausal symptoms: a theoretical synthesis of recent research. Women Health. 1988; 14(2): 105–27. PubMed Abstract \| Publisher Full Text\n\nKermanshahi S, Hosseinzadeh S, Alhani F: The effect of the group counseling program on the status of primary dysmenorrhea, dietary condition and exercise in Shahreyar Girl's High School. ZUMS Journal. 2009; 16(65): 49–60. Reference Source\n\nEzbarami S, Mirzaei B, Esfarjani F: Comparison the prevalence and severity of dysmenorrhea among athletes and non-athletes and its relation with body composition. Arak Medical University Journal. 2014; 16(11): 80–89. Reference Source\n\nMayo JL: A healthy menstrual cycle. Clin Nutr Insights. 1997; 5(9): 1–8. Reference Source\n\nSmith RP: Netter’s obstetrics and gynecology. Elsevier Health Sciences. 2008. Reference Source\n\nHaidari F, Akrami A, Sarhadi M, et al.: Prevalence and severity of primary dysmenorrhea and its relation to anthropometric parameters. Journal of hayat. 2011; 17(1): 70–7. Reference Source\n\nHarlow SD, Park M: A longitudinal study of risk factors for the occurrence, duration and severity of menstrual cramps in a cohort of college women. Br J Obstet Gynaecol. 1996; 103(11): 1134–42. PubMed Abstract \| Publisher Full Text\n\nUnsal A, Ayranci U, Tozun M, et al.: Prevalence of dysmenorrhea and its effect on quality of life among a group of female university students. Ups J Med Sci. 2010; 115(2): 138–45. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChung YC, Chen HH, Yeh ML: Acupoint stimulation intervention for people with primary dysmenorrhea: systematic review and meta-analysis of randomized trials. Complement Ther Med. 2012; 20(5): 353–63. PubMed Abstract \| Publisher Full Text\n\nAbadi Bavil D, Dolatian M, Mahmoodi Z, et al.: Dataset 1 in: A comparison of physical activity and nutrition in young women with and without primary dysmenorrhea. F1000Research. 2017. Data Source" }	[ { "id": "29899", "date": "23 Jan 2018", "name": "Narjes Bahri", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI read the paper carefully and find it good and suitable for publication. Only one point in my opinion needs to be clarified before publication as following: The two group of study have named \"case and control\" although the type of study has mentioned:\" comparative descriptive study\". There is a conflict that must be clarified.\nRecommendations for authors:\nThe type of study would be changed to: cross sectional study The name of groups would be change to: with dysmenorrhea / and without dysmenorrhea\nI hope my recommendation can improve the quality of the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "30514", "date": "07 Feb 2018", "name": "Mark Jones", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article requires additional information to be included. Also some of the information provided needs clarification.\nIn the abstract it is reported that dysmenorrhea is seen in almost 50% of women but in the introduction it is reported that overall prevalence is 60-90% in adolescent girls. This seems contradictory?\nThe sentences in the abstract results “Physical activity was calculated by MET scale (minutes/week). This index measured the amount of consumed energy at the time of activity relative to that consumed at resting time” would fit better in the abstract methods.\nIn the abstract please say how many of the 250 girls were in each comparison group.\nIt appears that the cases had greater physical activity than the controls (cases: 5518.75 vs controls: 4666.42). Please clarify and interpret accordingly.\nIn the abstract conclusion it says: “A healthier and more favorable nutrition style and more regular physical activity reduces the severity of dysmenorrhea in girls.” But the comparison is between cases and controls rather than a study of the effect of physical activity and nutrition on the severity of dysmenorrhea? Also this conclusion implies a causal effect but this study is a cross-sectional study of association?\nIt mentions “proper nutrition” in the abstract conclusion. Does this mean some of the study participants were not getting proper nutrition?\nWhat were the actual questions used to determine exposure status (i.e. dysmenorrhea)?\nMore information is required on the sample of participants included in the study. How many students were approached? How many participated? What were the reasons for any exclusions? How were the controls matched? The inclusion criteria for cases is not clear e.g. what does “painless (0 to 3) mean? What were the inclusion criteria for controls?\nWhat were the actual parameters assumed for the sample size calculation? E.g. what effect size was assumed?\nI could not follow the methods on how socioeconomic status and nutrition scores were obtained? Please clarify.\nIn the data analysis section please clarify which test was used for which data.\nWere all the potential confounders included in the analysis? E.g. what about smoking, alcohol, and other drug use?\nIn Table 2 I couldn’t understand what “3182/03” and “1930/12” meant? The education result was not included in table 2? Was there any missing data?\nWhy were the variables reported in Table 3 included in the logistic regression? Did you use a multivariable model? Why is the result for nutrition score no longer “statistically significant”?\nPlease interpret the main findings of the study in terms of clinical significance.\nPlease include a section on limitations of the study including missing confounding variables, potential for selection bias, potential for exposure/outcome misclassification.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "37383", "date": "29 Aug 2018", "name": "Zeinab Hamzehgardeshi", "expertise": [ "Reviewer Expertise midwifery", "women health", "sexual and reproductive health" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe topic is very attractive. Dysmenorrhea is a common gynecological pain among young women.The healthy life style can reduce the severity of pain in the young women. This cross sectional study is well designed. The instruments were valid. The results can be helpful for designing an intervention study.\nThe manuscript is suitable for indexing after the response to reviewers. Please revise based on the following comments:\nTitle: Indicate the study's design.\nIntroduction:\nPlease present previous studies in the field and clarify the research gap. Please present the aim of the study (state the specific objectives) at the end of the discussion.\nMethods:\nPlease clarify the validity and reliability of the Persian version of the questionnaires. Please describe the setting and location of the study. Please give the eligibility criteria, the sources, and the methods of selection of the participants. Please clearly define all variables. Describe any efforts to prevent potential bias.\nDiscussion:\nPlease discuss limitations of the study. Please clarify the generalisability of the study results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-59
https://f1000research.com/articles/6-2097/v1	06 Dec 17	{ "type": "Research Article", "title": "Effects of stress or infection on rat behavior show robust reversals due to environmental disturbance", "authors": [ "Samira Abdulai-Saiku", "Akshaya Hegde", "Ajai Vyas", "Rupshi Mitra", "Samira Abdulai-Saiku", "Akshaya Hegde", "Ajai Vyas" ], "abstract": "Background: The behavior of animals is intricately linked to the environment; a relationship that is often studied in laboratory conditions by using environmental perturbations to study biological mechanisms underlying the behavioral change. Methods: This study pertains to two such well-studied and well-replicated perturbations, i.e., stress-induced anxiogenesis and Toxoplasma-induced loss of innate fear. Here, we demonstrate that behavioral outcomes of these experimental manipulations are contingent upon the ambient quality of the wider environment where animal facilities are situated. Results: During late 2014 and early 2015, a building construction project started adjacent to our animal facility. During this phase, we observed that maternal separation stress caused anxiolysis, rather than historically observed anxiogenesis, in laboratory rats. We also found that Toxoplasma infection caused an increase, rather than historically observed decrease, in innate aversion to predator odors in rats. Conclusion: These observations suggest that effects of stress and Toxoplasma are dependent on variables in the environment that often go unreported in the published literature.", "keywords": [ "anxiety", "fear", "construction", "housing environment", "replicability" ], "content": "Introduction\n\nMultiple laboratories have reported that stress causes anxiogenesis in rats1–4. Similarly, well-replicated studies indicate that infection of rats with protozoan Toxoplasma gondii reduces innate aversion to predator odor5–11. This report describes our serendipitous observations that the direction for both behavioral changes is intricately dependent on the broader environment where animal facilities are situated.\n\nThe primary aim of our experiments was to study proximate mechanisms of anxiogenesis and innate aversion in rats. We used routine paradigms of maternal separation and Toxoplasma gondii infection that cause anxiogenesis and loss of innate aversion, respectively. However, construction of a building was initiated during the experiment adjacent to the animal holding facility. Results from this quasi-experimental change provided us with an unplanned opportunity to study the effects of change in environment on rat anxiety and defensive behaviors.\n\n\nMethods\n\nAdult male and female Wistar Han rats (7 to 8 weeks at the start of the experiments) were procured from InVivos, Singapore. Rats were housed in groups of two per cage (males and females were housed separately) with ad libitum access to food and water (24–26°C; 60–70% relative humidity; 12h light-dark cycle with lights on at 0700h). For all tests, animals were allocated to groups in a random manner. Experiments were conducted by SA-S and AHN who were blind to group allocations. Analysis was done by AV who was also blind to group allocations. All procedures were approved by the Institutional Animal Care and Use Committee of the Nanyang Technology University. All efforts were made to ameliorate any suffering of animals. None of our procedure involved induction of sustained pain requiring pharmacological interventions. Animals were observed daily to confirm lack of sickness related behaviors and weighed weekly. The behavior tests do not involve any use of shock or other painful stimuli. The dose of parasites used in this study does not result in weight loss or sickness behavior in this strain of rats.\n\nAt the end of all experiments, animals used in the Toxoplasma infection paradigm were sacrificed by decapitation and their brains were removed and flash frozen. In the case of the stress paradigm, animals were sacrificed by cardiac perfusion using cold phosphate buffered saline (PBS) followed by cold 4% paraformaldehyde.\n\nFemale rats were either injected with tachyzoites of type 2 Prugniaud strain of Toxoplasma gondii (5×106 tachyzoites in 500 µl phosphate buffered saline, i.p.;) or mock injected with the buffer alone between 2pm and 4pm. Parasites needed for the infection were maintained in vitro in human foreskin fibroblast cultures and were harvested using syringe lysis. Behavioral experiments were conducted seven weeks post-infection; a time-window consistent with chronic phase of the infection.\n\nAversion to cat odor was quantified in two different manners. For each run of the experiment, there was one control group and one Toxoplasma-infected group. Fifteen (15) animals were used in total for experiment 1 (8 control, 7 infected) and 19 animals were used in total for experiment 2 (10 control, 9 infected).\n\nAversion was first quantified in a rectangular arena with two opposite and identical arms (76 × 9 cm each), separated by a central part (9 × 9 cm in size; white Perspex). Animals were habituated to the arena for three consecutive days for 20 minutes each day. On the subsequent day, cat odors were presented in one bisect of the maze (1 ml each; bobcat urine from Maine Outdoor Solutions, USA). Animals were placed in the center of the maze and exploration time in both bisects of the arena was measured for 20 minutes. Trials were video recorded with offline analysis conducted using AnyMaze (Stoelting, USA). In this batch of animals, each received 500 µl of buffered saline intraperitoneally thirty minutes before the behavioral test.\n\nAversion to cat odor was also quantified in a circular arena that was arbitrally divided into four quadrants. Animals were habituated to the arena for three consecutive days for 20 minutes each day. On the subsequent day, cat odor, vanilla essence, water and the bedding from the animal’s home cage were presented in each quadrant of the maze. Animals were placed in the center of the maze and exploration time in all quadrants of the arena was measured for 20 minutes. Trials were video recorded with offline analysis conducted using AnyMaze (Stoelting, USA).\n\nEight week old breeders obtained from InVivos were allowed to acclimatize for at least 5 days before setting up breeding pairs (one male and one female per cage). Breeding cages were changed once a week as per normal, but with gentle handling of female, in case of pregnancy. Once pregnancy was certain (approx. 2 weeks), male was removed. 19 days after breeding pairs were set up (or if visually heavily pregnant), cages were checked daily for litters. Day of birth is assigned P0.\n\nMaternal separation was used as the stress model (P2-P14, daily). 16 animals were used in total; 8 stressed, 8 unstressed. On each of these days, the dam was removed from the cage and placed in a new cage with unsoiled bedding. Pups were then retrieved into another cage with unsoiled bedding, transported to a separate room and put on a heating pad for three hours every morning. At the end of the separation period, pups and then dam were sequentially returned to the original soiled cage. Also, soiled bedding was changed on postnatal day 2, 9 and 14; by returning pups to a clean cage that had been supplemented with a scoop of soiled bedding and nesting material from the original cage. This practice was repeated on postnatal day 18 if the bedding was considered significantly soiled in case of large litter sizes. Pups were weaned on postnatal day 21. Anxiety was quantified when the male pups reached adulthood (7–8 weeks of age). Anxiety was measured using home cage emergence assay (adapted from 12) and elevated plus-maze13.\n\nIn the home cage emergence assay, a rat placed in its home cage was transported to a well-lit room and habituated for five minutes. The cage was then left open by removal of the lid. The rat was offered a possibility of emerging from the home cage through a wire grid placed within the cage. The latency of emergence was recorded. Emergence was defined when all four limbs of the rat were placed on the grid. Trials were terminated at the emergence or at five minutes, whichever occurred earlier. Trials were video recorded and scored manually.\n\nThe elevated plus-maze consisted of a plus-shaped arena with two open (75 × 11cm, 1cm wall, 3–4 lux illumination) and two enclosed arms (75 × 11 cm, 26 cm wall). The arena was elevated to a height of 60 cm above the ground. The animal was placed at the center at the start of the trial. Exploration in open and enclosed arms was quantified for five minutes each.\n\nAll experiments for the stress paradigm were done using two groups of mice: stressed and unstressed.\n\nThe probability of type 1 error was calculated using unpaired two-tailed Student’s t-test. The standardized effect size was calculated using Cohen’s d14; with values above the magnitude of 0.8 interpreted as being of robust scale. Negative d values correspond to the comparisons where mean of experimental treatment was greater than that of respective controls. Mean inter-group difference was also calculated with 95% confidence intervals. Data is graphically presented as mean and standard error of the mean (SEM), along with individual values for each animal for each endpoint. Number of animals in each experimental group is noted in the figures. All statistical analysis was conducted using Graphpad Prism.\n\n\nResults\n\nIn the first set of animals, aversion to cat odor was quantified as percentage time in bisect containing cat odor relative to total trial duration. Rabbit odor was placed in the opposing bisect as a novel non-predator odor. Inter-group differences did not reach pre-determined threshold for statistical significance (Figure 1A; t13 = 1.78, p = 0.098). Despite the lack of sufficiently low type 1 error, the effect on mean was of robust magnitude (Cohen’s d = 0.949; Δ = -11.61% with 95% confidence intervals -25.68 to 2.46%). The maximum of animals from the infected group was below the median of the control animals. The robust effect size and the observation that infected mean was lower than controls in contrast to the multitude of published studies, led us to plan a further experiment to increase the statistical power.\n\nToxoplasma gondii-infected female rats showed increased aversion to bobcat odor in two sequential experiments (A and B). Ordinate depicts time spent by female rats chronically infected with Toxoplasma gondii near bobcat odor. Line graphs depict mean and standard error of the mean for control (black) and infected (gray) female rats. , p < 0.05; unpaired two-tailed Student’s t-test.\n\nIn this second set of animals, aversion to cat odor was quantified in a circular arena congruent to the initial design of reported infection effects. One quadrant contained soiled bedding from home cage of the animal, serving as the home base for exploratory sorties. Cat odor and a novel vanilla odor were placed in two adjoining quadrants. The ratio of time spent in cat quadrant relative to sum time spent in both cat and novel odor quadrants was calculated (chance = 50%). Toxoplasma infection, in contrast to earlier observations in the similar design, reduced percentage time spent near cat urine (Figure 1B; t17 = 2.70, p = 0.015). The effect of infection on innate aversion was of robust magnitude (Cohen’s d = 1.239; Δ = -16.46% with 95% confidence intervals -29.31 to -3.61%). The maximum of animals from the infected group was again observed to be below the median of the control animals.\n\nSerological examination confirmed that all animals in the infected groups sustained chronic infection with Toxoplasma gondii.\n\nAnimals subjected to early life maternal separation stress were tested in the elevated plus maze and home cage emergence test to determine the effect of maternal separation on anxiety behavior during adulthood.\n\nStressed animals, in contrast to earlier observations in a similar design, exhibited significantly less anxiety compared to unstressed controls. This was evident as increased percentage entries into anxiogenic open arms of elevated plus-maze (Figure 2A; t14 = 4.21, p = 0.0009). Stress-induced anxiolysis was of robust magnitude (Cohen’s d = -2.104; Δ = 27.77% with 95% confidence intervals 13.62 to 41.92%). The minimum of animals from the stressed group was higher than all but one animal from the unstressed group. Experimental treatment did not cause significant differences in number of entries made into non-anxiogenic enclosed arms of the maze (t14 = 1.98, p = 0.07). To preclude effects of entries in enclosed arms on open arm exploration, we further conducted a univariate analysis of variance for percentage open arm entries while employing number of enclosed entries as a covariate. This analysis revealed a significant increase in open arm exploration due to the stress independent of inter-group differences in enclosed arm entries (F1,13 = 14.898, p = 0.002). This is congruent with significant increase in number of head dips made during the trial by stressed animals (t14 = 3.41, p = 0.0042; Δ = 13.25 with 95% confidence intervals 4.94 to 21.56).\n\nOrdinate depicts number of entries into the open arm relative to total entries in open and enclosed arms of the elevated plus maze (A) and latency to emerge from the home cage into a novel environment (B). Line graphs depict mean and standard error of the mean for unstressed (black) and stressed (gray) male rats. , p < 0.05; **, p < 0.01; unpaired two-tailed Student’s t-test.\n\nStress-induced anxiolysis was also confirmed by home cage emergence test. In this assay, anxiolysis manifests as reduced latency to emerge into a novel environment from home cage. Stress significantly decreased the latency of home cage emergence (Figure 2B; t14 = 3.14, p = 0.0072). Stress-induced anxiolysis was also of robust magnitude in this assay (Cohen’s d = -1.57; Δ = -121.4s with 95% confidence intervals -204.2 to -38.5s). The maximum latency of animals from the stressed group was lower than median latency from the unstressed group.\n\n\nDiscussion\n\nExperimental treatment in the present report caused robust effects, as evidenced by substantial effect size and clear departure of mean differences from the chance. The direction of these effects is in stark contrast to those observed in previous reports1,6–8,15–17. For example, multiple experiments in several laboratories indicate that chronic Toxoplasma gondii infection causes loss of innate fear to predator odor in male and female rats6–8,11. Data in the present report, however, argue for a significant increase in innate fear post-infection. Similarly, stress-induced anxiogenesis has been reported across several laboratories and several paradigms. The current dataset, in contrast, exhibits significant anxiolysis post-stress. The cause of this discrepancy cannot be ascertained with confidence. In fact, we have observed stress-induced anxiogenesis and the infection-induced loss of fear in the same animal facility and same animal strain before these experiments1,5–7,18. The only difference between the experimental circumstances has been a construction project that was ongoing during the present experiments. The construction started across the road from the animal facility after our preceding baselines were conducted and during the present period of the behavioral testing. In fact, we observed reversal to Toxoplasma-induced loss of fear in female rats in experiments conducted in the animal facility after the cessation of building construction. It remains unclear if the effects of construction related to the change in ambient vibrational environment or some hitherto unknown variable. Although the acoustic noise in frequency range audible to humans remained unchanged during the period, we are not confident that the construction did not change the acoustic environment in sub-audible frequencies. It is interesting that the effects observed here do not correspond to a simplistic notion of greater baseline stress during the period. Effects of stress on anxiety are often presented to have an inverse U kind of reaction norm, whereby increasing stress enhances its effects on the behavioral and health parameters19–22. We observed an anxiolysis by experimental stress rather than greater anxiogenesis due to the accumulative stress of the treatment and environmental change. Thus, the present observations reiterate the often complex interactions between environment and behavior that could impose significant bounds on the interpretation of laboratory experiments. Related to this, same transgenic mice are known to exhibit divergent behavioral phenotypes across three experimental locations despite careful alignment of experimental protocols23.\n\n\nConclusions\n\nOften, unforeseen changes in the environment near animal facilities can significantly alter the direction of experimental effects in rodent research. This highlights the crucial role of often unreported and unquantified environmental context in the interpretation and replicability of the behavioral data.\n\n\nData availability\n\nDataset 1: Cat odour avoidance assay. Percentage time spent exploring the cat odour stimulus by control and Toxoplasma-infected rats in both experiment 1 and 2. 10.5256/f1000research.13171.d18632724\n\nDataset 2: Elevated plus maze anxiety test. Escape latency and percentage open arm entries for stressed and unstressed animals. 10.5256/f1000research.13171.d18632825", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was financially supported by Ministry of Education, Singapore (grant RG136/15 and RG 46/12).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nAshokan A, Hegde A, Mitra R: Short-term environmental enrichment is sufficient to counter stress-induced anxiety and associated structural and molecular plasticity in basolateral amygdala. Psychoneuroendocrinology. 2016; 69: 189–96. PubMed Abstract \| Publisher Full Text\n\nHillerer KM, Neumann ID, Slattery DA: From stress to postpartum mood and anxiety disorders: how chronic peripartum stress can impair maternal adaptations. Neuroendocrinology. 2012; 95(1): 22–38. PubMed Abstract \| Publisher Full Text\n\nMaes M, Song C, Lin A, et al.: The effects of psychological stress on humans: increased production of pro-inflammatory cytokines and a Th1-like response in stress-induced anxiety. Cytokine. 1998; 10(4): 313–18. PubMed Abstract \| Publisher Full Text\n\nPawlak R, Magarinos AM, Melchor J, et al.: Tissue plasminogen activator in the amygdala is critical for stress-induced anxiety-like behavior. Nat Neurosci. 2003; 6(2): 168–74. PubMed Abstract \| Publisher Full Text\n\nHari Dass SA, Vyas A: Toxoplasma gondii infection reduces predator aversion in rats through epigenetic modulation in the host medial amygdala. Mol Ecol. 2014; 23(24): 6114–22. PubMed Abstract \| Publisher Full Text\n\nVyas A, Kim SK, Giacomini N, et al.: Behavioral changes induced by Toxoplasma infection of rodents are highly specific to aversion of cat odors. Proc Natl Acad Sci USA. 2007; 104(15): 6442–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdulai-Saiku S, Vyas A: Loss of predator aversion in female rats after Toxoplasma gondii infection is not dependent on ovarian steroids. Brain Behav Immun. 2017; 65: 95–8. PubMed Abstract \| Publisher Full Text\n\nBerdoy M, Webster JP, Macdonald DW: Fatal attraction in rats infected with Toxoplasma gondii. Proc Biol Sci. 2000; 267(1452): 1591–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebster JP: The effect of Toxoplasma gondii on animal behavior: playing cat and mouse. Schizophr Bull. 2007; 33(3): 752–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWebster JP: Rats, cats, people and parasites: the impact of latent toxoplasmosis on behaviour. Microbes Infect. 2001; 3(12): 1037–45. PubMed Abstract \| Publisher Full Text\n\nWebster JP, Lamberton PH, Donnelly CA, et al.: Parasites as causative agents of human affective disorders? The impact of anti-psychotic, mood-stabilizer and anti-parasite medication on Toxoplasma gondii's ability to alter host behaviour. Proc Biol Sci. 2006; 273(1589): 1023–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVan den Hove DL, Blanco CE, Aendekerk B, et al.: Prenatal restraint stress and long-term affective consequences. Dev Neurosci. 2005; 27(5): 313–20. PubMed Abstract \| Publisher Full Text\n\nWalf AA, Frye CA: The use of the elevated plus maze as an assay of anxiety-related behavior in rodents. Nat Protoc. 2007; 2(2): 322–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen J: Statistical power analysis for the behavioral sciences. Hilsdale. NJ: Lawrence Earlbaum Associates. 1988; 2. . Reference Source\n\nGolcu D, Gebre RZ, Sapolsky RM: Toxoplasma gondii influences aversive behaviors of female rats in an estrus cycle dependent manner. Physiol Behav. 2014; 135: 98–103. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLadd CO, Huot RL, Thrivikraman KV, et al.: Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog Brain Res. 2000; 122: 81–103. PubMed Abstract \| Publisher Full Text\n\nLee RS, Sawa A: Environmental stressors and epigenetic control of the hypothalamic-pituitary-adrenal axis. Neuroendocrinology. 2014; 100(4): 278–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoe AS, Ashokan A, Mitra R: Short environmental enrichment in adulthood reverses anxiety and basolateral amygdala hypertrophy induced by maternal separation. Transl Psychiatry. 2016; 6(2): e729. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRusso SJ, Murrough JW, Han MH, et al.: Neurobiology of resilience. Nat Neurosci. 2012; 15(11): 1475–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSapolsky RM: Stress and the brain: individual variability and the inverted-U. Nat Neurosci. 2015; 18(10): 1344–6. PubMed Abstract \| Publisher Full Text\n\nMendl M: Performing under pressure: stress and cognitive function. Applied Animal Behaviour Science. 1999; 65(3): 221–44. Publisher Full Text\n\nAshokan A, Sivasubramanian M, Mitra R: Seeding Stress Resilience through Inoculation. Neural Plast. 2016; 2016: 4928081. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrabbe JC, Wahlsten D, Dudek BC: Genetics of mouse behavior: interactions with laboratory environment. Science. 1999; 284(5420): 1670–2. PubMed Abstract \| Publisher Full Text\n\nAbdulai-Saiku S, Hegde A, Vyas A, et al.: Dataset 1 in: Effects of stress or infection on rat behavior show robust reversals due to environmental disturbance. F1000Research. 2017. Data Source\n\nAbdulai-Saiku S, Hegde A, Vyas A, et al.: Dataset 2 in: Effects of stress or infection on rat behavior show robust reversals due to environmental disturbance. F1000Research. 2017. Data Source" }	[ { "id": "28765", "date": "11 Dec 2017", "name": "Jaroslav Flegr", "expertise": [ "Reviewer Expertise Evolutionary biology", "evolutionary parasitology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present interesting data showing that, most likely, an unknown environmental factor or factors can qualitatively modify the behavioral responses of experimental animals on various standard stimuli (here the maternal separation stress and the Toxoplasma infection), which could result in unexpected results of standard experiments. The methods are clear and with sufficient details described, and the collected data are analyzed, presented and interpreted in a proper way. The authors suggested that the most probable factor that influenced the outputs of their experiments was (acoustical or mechanical) disturbance from a building construction project that had started adjacent to their animal facility during their experiments. This explanation seems to be reasonable, however, it would be a little bit difficult (and expensive) to test its validity.\nI consider the results (and conclusions) of this study to be not only very interesting, but also very important. It is highly probable that the same or similar phenomena are frequently seen by many researchers, however, they are mostly considered to be just the results of some technical error – “This new student/technician is really terrible, he certainly confused the labels on the cages/test tubes!”. We can just hope that the publication of the present paper will have the “#MeToo effect” – that it will encourage other researchers to publish their own puzzling results.\nI agree that the inverted-U shape (or U-shape) relations between many physiological variables is mostly responsible for frequently observed opposite reaction of a biological system on the same stimuli. Under one situation (e.g., when no building construction project is going on) the background level of stress is low and adding some stress factor, e.g., infecting animals with Toxoplasma, will shift the behavioral response toward the maximum of the inverted-U function. Under another situation, when the background level of stress is higher (when the building construction project is going on) additional stress (e.g., the infection with Toxoplasma) will shift the behavioral response behind the maximum of the inverted-U function, which will result in an opposite behavioral reaction on the infection. In the review article on the methodological problems of studying the effects of toxoplasmosis using Toxoplasma-human model (Flegr, 2013), I showed that on genetically polymorphic outbred animals, including humans, the same factor often influences some individuals in one way and another individuals in an opposite way, depending on their (unknown) genotype. Very often, we can see that population means of the output variable in the affected individual and in the controls remains the same; however, variance of the output variable in the population of affected individuals grows significantly. For example, comparison of Cattell’s 16PF personality profiles of women showed that infected women had higher intelligence and lower guilt proneness than Toxoplasma-free women. At the same time, they differed in the variance of four other personality factors, namely protension, surgency, shrewdness and self-sentiment integration (Flegr and Havlíček, 1999). It is therefore very important to study the effects of particular factors not only on population mean of the output variable but also on the variance of this variable. We should never forget that the F-test (or a permutation test performed on squared Z- scores) is not just a pesky technique for testing presumptions of parametric statistical tests, but often it can also be an important and powerful tool for detecting biologically relevant effects of the factor under study.\nExactly the same mechanism can explain why males and females so often react to the same factor in an opposite way. In most animal species, males and females are not same. Therefore, many physiological parameters of males and females differ in their (mean) position in relation to the maximum of the inverted-U function. Consequently, they will respond to the same factor by the opposite-direction shifts. For example, 10 of 16 Cattell’s personality factors are shifted in an opposite direction in men and women in reaction to the Toxoplasma infection (Flegr et al., 2000; Flegr et al., 1996). Similarly, Toxoplasma-infected men rate the smell of highly diluted cat urine as more attractive while infected women rate this smell as less attractive than their non-infected peers (Flegr et al., 2011). It is worthwhile in the context of the present Abdyla-Saiku et al. article to mention that our recent study showed the very opposite pattern, namely higher attractiveness of the smell in the infected women and lower in the infected men, when undiluted cat urine was used as the stimulus (Flegr et al. 2017).\n\nBack to the present article. It can be published in its present form. I would just suggest that the authors cite the old study (Vyas et al., 2007) showing the inverted-U shaped response of infected rats on the smell of cat urine. When describing their experimental setup, the authors should better emphasize the fact that stressed mothers, not stressed pubs were used in all ethological tests. Authors should also double-check whether all Latin names of species and genera are printed in italic, both in the main text and in the References.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3307", "date": "16 Jan 2018", "name": "Rupshi Mitra", "role": "Author Response", "response": "We thank reviewer for suggestions and comments. This has helped us to improve this manuscript during the revision. We have now submitted version 2 of this manuscript to the F1000Research.In the version 2, we have included a discussion of non-monotonic response of Toxoplasma in the introduction. We would also like to clarify that we tested stressed pups not their mothers. Pups were maternally deprived before weaning, allowed to reach adulthood and then tested.This has now been made clear during the revision.We have carefully checked and corrected all Latin names." } ] }, { "id": "28761", "date": "19 Dec 2017", "name": "Terence Y Pang", "expertise": [ "Reviewer Expertise Rodent behaviour testing", "anxiety", "stress" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a well-written, clearly presented manuscript describing unexpected behavioural phenotypes of two well-established rodent models which routinely lead to rats having anxiolytic or anxiogenic behaviours. In the field of behavioural neuroscience where robustness of results and reproducibility is vital, the reporting of negative or contrary outcomes remains important. This report raises substantial concern for the rodent research occurring across that time period. It is crucial that universities and research institutes be educated on the impact that infrastructure development has on researchers, and the time and financial costs it imposes on research teams/projects.\n\nIn the Introduction, which is rather short, it would be useful to include one or two paragraphs referencing evidence that both Toxoplasma infection and maternal separation models are prone to environmental modification. One of the included references (Koe et al., Transl Psychiatry 2016) is an example of environmental modification of a robust maternal separation-induced adult phenotype. See also Sahafi E et al., Physiol Behav 2018 as another example of an external modifier of anxiety behaviour.\n\nThis manuscript is limited in that there is no molecular data to be paired with the interesting behavioural phenotype. A comparison of monoamine-relevant genes ala Récamier-Carballo S et al., Behav Pharmacol 2017 would have been ideal. But this can be speculated upon in the Discussion. Could also mention the involvement of environment-induced epigenetic changes, see McCoy CR et al., Eur J Neurosci 2016 : DNA methylation changes in the hippocampus.\n\nThe major shortcoming of this manuscript is that I am unsure about how one would go about quantifying structural disturbances. Is it based solely on the unexpected behavioural phenotype observed? Or has the phenotypes consistently shifted during the stated period before returning to “normal”? Have there been anecdotal accounts of construction noise in the rodent facility? Is there any data about building vibrations? (Civil engineers would have the equipment to measure structural vibrations).\n\nAssuming the significant external source of variability (as compared to a new experimenter who is a inexperienced at handling rodents and conducting the behavioural tests), it would be useful to include litter sizes and M/F sex ratios. Is there body weight data in the event that feeding behaviour was also altered?\n\nIt is unusual to only present EPM data as % entries in open arm. What about total time in open arms as a % of the test duration?\n\nIs it possible to include schematics of the different test arenas for Figure 1? Do the authors have habituation data (total time spent moving, distance travelled) for odor aversion tests? If the infected rats are anxious, they could be observed to display non- or lesser habituation even at baseline in the absence of a predator odor. If this was the case, it would only serve to strengthen the interpretation.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3308", "date": "16 Jan 2018", "name": "Rupshi Mitra", "role": "Author Response", "response": "We thank reviewer for suggestions and comments. This has helped us to improve this manuscript during the revision. We have now submitted version 2 of this manuscript to the F1000Research.Introduction has been modified in version 2 to include prior work showing that effects of Toxoplasma infection and maternal separation are subject to environmental modification (references 12 through 16 in the bibliography).We have also revised the discussion to include plausible proximate mechanisms including epigenetic changes and monoamines. Please see paragraphs immediately preceding the conclusions.We now return to the reviewer’s comment about ambivalent nature of quantifying structural disturbances. We have indeed observed return to stress-induced anxiogenesis and Toxoplasma-induced loss of fear once construction project abated. Toxoplasma effects were eventually published (reference 7 in the revised bibliography; DOI: 10.1016/j.bbi.2017.04.005). Same set of experimenters conducted experiments before, during and after the construction project. Thus, congruent stress- and infection- effects before and after construction project suggest that the environmental modification brought about by the construction explains atypical effects in the interim.Experimental groups were coded during the experiment. For example, in case of Toxoplasma infection, experimenter did not know infection status of the individual animals; and groups were merely identified with codes during the statistical analysis. Hence we did not notice the reversal until long after the experiment was over, data was analyzed and infection status was confirmed using serology. This precluded systematic investigation of the environmental variables during the period of experiment itself. Although the acoustic noise in frequency range audible to humans remained unchanged during the period, we are not confident that the construction did not change the acoustic environment in sub-audible frequencies. Similarly we did not have opportunity to measure structural vibrations as the project was finished while we were analyzing the data and confirming group assignments using serology.Toxoplasma gondii infection did not cause significant change in body weight of animals (179.1 ± 4.708, n = 8 for control; 183.1 ± 2.706, n = 7 for infected; p = 0.5). This information is now included in methods section of the revised manuscript. We have revised the manuscript to include date for percentage open arm time (p < 0.001) for EPM in figure 2. We have also included schematics of test arena in revised Figure 1. Please note that this has changed panel number for figures in the results and legends.Unfortunately, we did not record videos for habituation sessions. We have earlier shown that Toxoplasma infection does not affect locomotion or exploration in open field arena." } ] } ]	1	https://f1000research.com/articles/6-2097
https://f1000research.com/articles/7-54/v1	15 Jan 18	{ "type": "Method Article", "title": "Analysis of removal of cadmium by action of immobilized Chlorella sp. micro-algae in alginate beads", "authors": [ "Christian Valdez", "Yomaira Perengüez", "Bence Mátyás", "María Fernanda Guevara", "Christian Valdez", "Yomaira Perengüez", "María Fernanda Guevara" ], "abstract": "Cadmium (Cd) is a metal that can negatively interfere with the metabolic systems of living beings. The objective of this work was to evaluate the capacity for cadmium removal in aqueous solutions by immobilized Chlorella sp. in calcium alginate beads. Beads without Chlorella sp. were used as a control. All the treatments were established in triplicate for 80 min, at four concentrations of cadmium (0, 20, 100 and 200 ppm), taking samples of aqueous solution every 10 min, to be read using atomic absorption equipment. The study determined that the treatment of alginate beads with immobilized Chlorella sp. removed 59.67% of cadmium at an initial concentration of 20 ppm, this being the best removal result.", "keywords": [ "Chlorella sp.", "cadmium removal", "immobilization", "alginate" ], "content": "Introduction\n\nPollution from the use of metals as a consequence of waste generated by industries is a constant concern, since they may end up being transferred to the environment1,2. The need to develop technologies for the remediation of water contaminated with cadmium should be considered as a priority in Ecuador. The objective of this study was to evaluate the capacity for cadmium removal in aqueous solutions by immobilized Chlorella sp. in calcium alginate beads. This study used the micro-algae Chlorella sp., for the bio-removal of cadmium, since the use of cells of Chlorella sp. immobilized in beads of alginate has been successfully exploited3 and its potential use has been established in the bioremediation of contaminants such as metals4, nutrients5 and other industrial pollutants6.\n\n\nMethods\n\nThe assay was carried out with aqueous solutions of cadmium. This solution was placed together with the alginate beads in Florence flasks. Every 10 minutes a sample of the aqueous solution was taken (10 mL approximately) with a glass pipette, and each sample was placed in a test tube. The concentration of metal was measured by aspiration atomic absorption spectrophotometer.\n\nTo make the control beads, the procedure described in a previous work was followed7. Briefly, 2 grams of sodium alginate (Loba Chemie, high density) were dissolved in 200 mL of ultra-pure water to obtain a homogeneous mixture, adjusting the pH between 7.8–8.0. pH was adjusted by addition of 0.1 molar sodium hydroxide or 0.1 molar hydrochloric acid, in sodium alginate paste (homogeneous mixture). One drop at a time was slowly poured into a 1% w/v calcium chloride solution with constant stirring.\n\nFor the immobilization of Chlorella sp. in alginate beads, liquid cultures were used. The volume of culture to be used was established to reach a concentration of 25 X 106 cells mL per each mL of alginate. For the preparation of the beads, 2 g of sodium alginate were dissolved in microalgae culture, as above.\n\nExperimental liquids were placed in 12 Florence Flasks of 500 mL capacity, inside which were placed 50 g of alginate beads without immobilized Chlorella sp. and 200 mL of cadmium solution at different concentrations (0, 20, 100 and 200 ppm). Each concentration was performed in triplicate, essay was evaluated during 80 minutes, aqueous solution samples were taken at 10 min intervals. Each Florence flask was placed on a shelf in the laboratory, the location of each bead on this surface was performed randomly. The flasks were subjected to constant aeration with air pressure of 0.012 MPa for 80 min. Every 10 minutes, an aqueous solution sample was taken to measure the concentration of metal present. These were the control treatments.\n\nFor the experimental samples, 50 g of alginate beads with immobilized Chlorella sp. were placed in each flask with the same concentrations of cadmium solution as the controls. These experiments were also performed in triplicate. These experimental samples were used to establish whether the addition of Chlorella sp. in the beads, increases or decreases the alginate removal activity.\n\nThe determination of metal concentration in the solutions was measured with a VARIAN direct aspiration atomic absorption spectrophotometer (VARIAN. Model: SpectrAA 55, Wavelength: 326.1 nm), using air-acetylene flame with cadmium lamp, and results are presented as % of removal of cadmium.\n\nFor viability tests, color change of the cultures exposed to cadmium was evaluated. For this purpose 100 mL of liquid Chlorella cultures were used (25 × 106 cells/mL) and they were subjected to different concentrations of Cd (0, 20, 100 and 200 ppm), in triplicate. The color change was monitored at the first hour of treatment, at 24 hours and at 7 days.\n\n\nResults and discussion\n\nColor changes were observed in the solutions (Figure 1, Dataset 1 and Dataset 2) of free micro-algae cells exposed to different concentrations of cadmium, which is a product of the toxicity of the metal, while the solutions without the presence of metal maintained a green color, characteristic of Chlorella sp.7\n\nsubjected to different concentrations of Cd, at 60 minutes, (A) 0 ppm Cd, (B) 20 ppm Cd, (C) 100 ppm Cd, (D) 200 ppm Cd. Green color means that algae remain viable, brown color means that Chlorella loses viability.\n\nIt was determined that Chlorella sp. exposed to a concentration of 20 ppm cadmium at 60 minutes of contact does not react, and shows no noticeable changes in coloration. On the other hand, the micro-algae did not show resistance to concentrations 100 and 200 ppm of Cd, revealing that the cells did not remain viable due to the color variation and high toxicity of the metal used, going from green to a grayish brown in the first 60 minutes of the start of the trial. These results show to decrease algal growth and inhibit photosynthesis8 100 ppm or higher.\n\nIt is verified that the best treatment for Cd removal is the one corresponding to alginate beads with immobilized Chlorella sp. at the concentration of 20 ppm of Cd at 80 min. It was determined that at low metal concentrations the micro-algae enhances its removal capacity achieving a removal percentage of 59.67% with Chlorella sp., being significantly higher (p value <0.001) than the removal presented without Chlorella sp. (55.56%), explained by the viability process that showed that Chlorella sp. can withstand the toxicity of the metal for periods of at least 60 min at concentrations of 20 ppm (Figure 2 and Figure 3). As demonstrated by 9, micro-algae work better at lower concentrations of metal.\n\nChlorella sp. immobilized in alginate beads could be used for bioremediation processes of cadmium at low concentrations of the metal, since the presence of viable biomass of the micro-algae potentiates the removal capacity of the alginate. Non-viable Chlorella sp., because of the high concentration of cadmium (100 and 200 ppm), decreases its removal capacity, yielding better results in beads treated without Chlorella sp. Thus, the alginate matrix without micro-algae could be used effectively in Cd removal processes at high metal concentrations.\n\n\nData availability\n\nDataset 1. Percentage of Cd in aqueous solution at 80 minutes. DOI, 10.5256/f1000research.13527.d19026610\n\nDataset 2. Raw data for the percentage removal of cadmium with and without Chlorella alginate beads at all concentrations and triplicate experiments DOI, 10.5256/f1000research.13527.d19028111", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBallesteros JL, Larenas C: Determinación de la eficacia de Azolla caroliniana como matriz de hiperacumulación de metales pesados cuantificados. Repositorio isntitucional UPS. 2012. Reference Source\n\nPlaza J: Remoción de metales pesados empleando algas marinas. Repositorio UNLP. 2012. Reference Source\n\nMartínez M, García M: Aplicaciones ambientales de microorganismos inmovilizados. Scielo. 2011.\n\nBayramoğlu G, Tuzun I, Celik G, et al.: Biosorption of mercury(II), cadmium(II) and lead(II) ions from aqueous system by microalgae Chlamydomonas reinhardtii immobilized in alginate beads. Int J Miner Process. 2006; 81(1): 35–43. Publisher Full Text\n\nde-Bashan LE, Moreno M, Hernandez JP, et al.: Removal of ammonium and phosphorus ions from synthetic wastewater by the microalgae Chlorella vulgaris coimmobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense. Water Res. 2002; 36(12): 2941–2948. PubMed Abstract \| Publisher Full Text\n\nRehman A, Shakoori AR: Heavy metal resistance Chlorella spp., isolated from tannery effluents, and their role in remediation of hexavalent chromium in industrial waste water. Bull Environ Contam Toxicol. 2001; 66(4): 542–547. PubMed Abstract \| Publisher Full Text\n\nArias A: Análisis de remoción de cromo por acción de la microalga Chlorella sp. inmovilizada en perlas de alginato. (tesis de pregrado). Universidad Politécnica Salesiana,Quito, Ecuador. 2017. Reference Source\n\nEl-Naggar AH, El-Sheekh MM: Abolishing cadmium toxicity in Chlorella vulgaris by ascorbic acid, calcium, glucose and reduced glutathione. Environ Pollut. 1998; 101(2): 169–74. PubMed Abstract \| Publisher Full Text\n\nShanab S, Essa A, Shalaby E: Bioremoval capacity of three heavy metals by some microalgae species (Egyptian Isolates). Plant Signal Behav. 2012; 7(3): 392–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nValdez C, Perengüez Y, Guevara MF, et al.: Dataset 1 in: Analysis of removal of cadmium by action of immobilized Chlorella sp. microalgae in alginate beads. F1000Research. 2018. Data Source\n\nValdez C, Perengüez Y, Guevara MF, et al.: Dataset 2 in: Analysis of removal of cadmium by action of immobilized Chlorella sp. microalgae in alginate beads. F1000Research. 2018. Data Source" }	[ { "id": "29838", "date": "23 Jan 2018", "name": "Juan Enrique Tacoronte Morales", "expertise": [ "Reviewer Expertise Applied chemical ecology and applied organic chemistry" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe problems of pollution with heavy metals is a serious environmental issue. The use of microalgae supported on different substrates is an eco-biotechnological variant of great importance in sustainable conditions. The work is clear, concise and develops a simple and applicable strategy in field and laboratory conditions. The utilization of alginate beds (Ca 2+) is a great technological solution", "responses": [] }, { "id": "29834", "date": "24 Jan 2018", "name": "Bernadett Gálya", "expertise": [ "Reviewer Expertise Environmental technologies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn my opinion, this article is well editable and easy to follow. The scope of each chapter is appropriate. In the Introduction, they briefly presented the problem behind the study. The study design is appropriate and the work is technically sound. They cited 11 references which is strongly connected to this topic. These are from 1998 to 2018, all in all, current literature was used. The method was described in detail, thus it is easy to replicate. They created three main parts in the methods. In my opinion is fine, but I suggest that the third subchapter should be the test used during the experiment. Otherwise, the materials, repetition and measurement which they are used are appropriate. The charts that are used are clear and easy to follow these in the text. In this article, the statistical analyses is applicable. May I suggest, that in the future they should use some other statistical test to compare different treatments (such as Tukey or Duncan-test). All source data are underlying the results available to ensure full reproducibility. The conclusions are drawn adequately supported by the results. Summary, this article is clear, easy to follow, used appropriate methods and scientifically based. Thus, it will be very useful to solve the examined problem.", "responses": [] } ]	1	https://f1000research.com/articles/7-54
https://f1000research.com/articles/4-633/v1	27 Aug 15	{ "type": "Research Article", "title": "Pre-hospital delay in Vietnamese patients hospitalized with a first acute myocardial infarction: A short report", "authors": [ "Hoa L. Nguyen", "Dat T. Phan", "Duc A. Ha", "Quang N. Nguyen", "Robert J. Goldberg", "Dat T. Phan", "Duc A. Ha", "Quang N. Nguyen", "Robert J. Goldberg" ], "abstract": "Background Administration of coronary reperfusion therapy to patients with an acute myocardial infarction (AMI) within the proper timeframe is essential in avoiding clinical complications and death. However, the extent of pre-hospital delay is unexplored in Vietnam. This report aims to describe the duration of pre-hospital delay of Hanoi residents hospitalized with a first AMI at the Vietnam National Heart Institute. Methods A total of 103 Hanoi residents hospitalized at the largest tertiary care medical center in the city for first AMI, who have information on prehospital delay was included in this report.Results One third of the study sample was women and mean age was 66 years. The mean and median pre-hospital delay duration were 14.9 hours and 4.8 hours, respectively. The proportion of patients who delayed <6 , 6-<12, and ≥ 12 hours were 45%, 13%, and 42%, respectively.Conclusions Our data shows that a prolonged pre-hospital delay is often observed in patients with a first AMI in Vietnam. In order to confirm these findings, a full-scale investigation of all Hanoi residents hospitalized with first AMI is needed. Increasing public awareness about AMI treatment is vital in encouraging patients to seek medical care timely after experiencing AMI symptoms such that received treatment is most effective.", "keywords": [ "Acute myocardial infarction", "pre-hospital delay", "outcomes", "epidemiology", "Vietnam" ], "content": "Introduction\n\nVietnam is a low-middle income country, and has been in the midst of an important epidemiological transition. Over the past two decades, the overall morbidity and mortality rates resulting from non-communicable diseases (NCDs) has been rising rapidly and presents itself as a major problem in Vietnam. One quarter of all deaths annually in Vietnam are now the result of cardiovascular diseases, making it the leading cause of death.\n\nTimely administration of reperfusion therapy to patients with an evolving acute myocardial infarction (AMI) is of utmost importance in reducing clinical complications and death. Previous research studies have convincingly proved that reperfusion treatment is most effective if patients with ST-segment elevation myocardial infarction (STEMI) are treated in a timely fashion, particularly within one hour of acute symptom onset1,2; the relation between extent of pre-hospital delay and outcomes after non-STEMI has not been firmly established.\n\nDespite the importance of prolonged pre-hospital delay on the timely receipt of effective treatments and short-term outcomes, so far we have found no evidence of studies conducted to examine the extent of pre-hospital delay among adult patients hospitalized with AMI in Vietnam. This short report hence aims to describe the extent of pre-hospital delay in Hanoi residents who were non-transferred, hospitalized with a first AMI at the Vietnam National Heart Institute in 2010.\n\n\nMethods\n\nThis study was approved by the Institutional Review Board at the Institute of Population, Health and Development. A waiver of patient consent was approved by the Institutional Review Board. All patients’ personal information was de-identified before analysis.\n\nThis study was conducted at the Vietnam National Heart Institute (VNHI)3,4. This hospital is a 250 bed tertiary care medical center in Hanoi (2009 census = 6.5 million) which manages the majority of Hanoi residents hospitalized with acute coronary disease and other NCDs.\n\nComputerized printouts of discharged patients from the VNHI in 2010 with possible AMI were collected and International Classification of Disease codes for possible AMI (I20–I25) were reviewed. Two cardiologists validated these cases utilizing predefined criteria developed by the World Health Organization. They include having a suggestive medical history, serum enzyme increasing above the hospital’s reference range, and serial electrocardiographic (ECG) findings during hospitalization consistent with the presence of AMI; at least two of these three criteria needed to be present for an AMI to have occurred5. There were 315 Hanoi residents, who satisfied the criteria for AMI were identified. However, 13 (4%) patients with history of AMI and 199 (66%) patients transferred from other facilities were removed from the analysis. Patients were then further divided into groups of STEMI and non-STEMI, utilizing standard classification techniques6. All ECGs of potential AMI patients were reviewed by a physician under the supervision of a senior cardiologist. Due to reviewed ECGs’ inadequate quality, nine patients with AMI could not be classified as STEMI or non-STEMI.\n\nFor all AMI-validated cases through the aforementioned process of independent adjudication utilizing eligibility criteria, information on social demographics, clinical, medical management and hospital discharge status were collected from medical record reviews and recorded onto a standardized case report form by trained study physicians.\n\nPre-hospital delay is the primary outcome investigated by this study. The delay time was defined as the time interval between the onset of symptoms suggestive of AMI and the patient’s arrival at medical centers7,8. Our trained physicians reviewed any information they could seek in medical records, which described the duration of pre-hospital delay from emergency personnel, nurses, and physicians notes. Information on pre-hospital delay was collected in minutes (as a continuous variable) for 63% (n=65) of patients. This variable was further categorized according to cut-points of 6 and 12 hours, which were commonly applied in prior studies, and based on the data distribution a. In 37% of patients (n=38) no concrete time of symptom onset was reported, only a delay time interval was recorded in patient’s medical record (e.g. <6, 6-<12, and ≥12 hours); these patients were incorporated into our analysis of delay time when it was constructed as a binary outcome.\n\nThe overall mean (standard deviation-SD) and median (inter quartile range-IQR) duration of pre-hospital delay was calculated according to standard methods. Data were shown as percentages for categorical variables and compared between patients who delayed ≥ 6 hours, and patients who sought medical care earlier, using chi-square or Fisher exact tests; medians (inter quartile range-IQR) for continuous variables were calculated and compared using the Wilcoxon sum rank tests. Since the sample size in this study was small, no regression analysis was performed. All analyses were performed using STATA 11.0 (StataCorp. TX).\n\n\nResults\n\nA total of 103 non-transferred Hanoi residents hospitalized with a first AMI at VNHI in 2010 were included in the report. The study sample had an average age of 66 years with a standard deviation of 13 years. One third of the study sample was women, and 69% were classified as STEMI.\n\nWithin the group of 65 patients reporting an exact time of symptom onset, the overall mean and median durations of pre-hospital delay were 14.9 hours (SD: 23 hours) and 4.8 hours (IQR: 3–10 hours), respectively (Figure 1). Among patients with STEMI (n=42), mean and median delay times were 16.4 hours (SD: 26 hours) and 4.8 hours (IQR: 3.0–6.2 hours), respectively. The proportion of patients who delayed <2 hours, 2-<4 hours, 4-<6 hours, 6-<12 hours, and ≥12 hours were 14%, 25%, 23%, 15%, and 23%, respectively.\n\nWhen combining data from these patients (n=65), with more non-specific data from patients who reported duration of delay in a time interval (n=38), the proportion of patients who delayed <6 hours, 6-<12 hours, and ≥12 hours were 45%, 13%, and 42%, respectively (Figure 2).\n\nPatients who had a delay time over 6 hours were more likely to be female compared to their counterparts, who had a delay time of less than 6 hours (Table 1).\n\nPre-hospital delay was defined as the duration from onset of acute symptoms suggestive of AMI to hospital arrival.\n\nP-values from chi square or Fisher exact tests for categorical variables, and t-tests or Wilcoxon-sum rank tests for continuous variables.\n\nSD: Standard deviation; IQR: Inter quartile range; STEMI: ST-segment elevation MI; LDL: Low-density lipoprotein; eGFR: estimated glomerular filtration rate\n\n*The Kinh people are the majority ethnic group in Vietnam, comprising 87% of the population (census 2009).\n\n†Missing data in 9 patients\n\n\nDiscussion\n\nThe results of this study indicated that Vietnamese patients experienced a considerable pre-hospital delay after the onset of AMI symptoms (median: 4.8 hours), with more than half experiencing a delay time of 6 hours or longer. Women delayed seeking care to a greater extent than men.\n\nThis report is, to our knowledge, the first in Vietnam to describe the duration of pre-hospital delays in patients hospitalized with a first AMI. In this study, patients experienced considerably longer pre-hospital delay compared with patients from both low-middle income countries and high-income countries7–16. For example, a survey of 250 patients hospitalized with AMI in Shanghai, China in 2010 reported that the median time for patients to seek treatment was around 2 hours alongside another one half hour (median time) for transportation9. Another recent survey from 270 patients hospitalized with AMI in Bandar Abbas, Iran found that approximately 20% of patients experienced a delay time of 6 hours or more13. A study of more than 200 patients hospitalized with acute coronary syndrome in London, England between 2001 and 2004 reported that the median pre-hospital delay time was 2 hours15. Prior studies in the U.S. have shown that patients hospitalized with AMI delayed, on average, approximately 2 hours following the onset of symptoms suggestive of AMI11,16. However, the duration of pre-hospital delay in our study was comparable to that reported from more than 60 African American patients hospitalized with AMI in the US in 2003–2004 (median: 4.3 hours)17 and from more than 400 patients hospitalized with AMI in Croatia in 2005 (median: 4.3 hours)18. We found that comparatively, female patients tended to seek treatment much later than male patients after the onset of suggestive AMI symptoms. This result is consistent with studies conducted previously9,10,19–22, but contradictory to others, which reported that there was no association between sex and pre-hospital delay12–14.\n\nOur findings emphasize the need to implement intervention programs to increase awareness of the general population with regards to the importance of seeking treatment timely after experiencing AMI suggestive symptoms. Prolonged delay may be associated with an individual’s risk of cardiac death, serious clinical complications, and delays in the receipt of effective treatments, primarily coronary reperfusion therapy. Moreover, to understand why these patients fail to react promptly to their AMI symptoms, focus group discussions and/or in-depth interviews should be carried out in patients hospitalized with AMI focusing on their levels of cognition, knowledge, and attitudes toward hospitals and health care. Additional studies are warranted in exploring the effects of educational attainment, insurance coverage, neighborhood characteristics, psychosocial aspects, and other factors that may serve as either facilitators or obstacles for patients to the timelier seeking of medical care, particularly of factors that may be amenable to modify.\n\nThere are several imitations in this study. First, our data was collected from only one hospital, the VNHI in Hanoi, which limits the generalizability of our results to patients hospitalized in other medical centers across Hanoi. Second, due to our small sample size, we failed to identify potential factors that might relate to the excessive delay time. Third, we were only able to calculate total duration of pre-hospital delay, were not able to calculate patient delay time and transportation delay separately since the time when patients made a treatment-seeking decision was not recorded in hospital medical records. In Vietnam, patients with signs and symptoms suggestive of AMI normally call for an ambulance or reach the hospital by themselves, and extremely few patients have a primary care doctor to consult. In terms of transportation time, this study included only patients who reside in Hanoi. Finally, due to the retrospective design of the study, we were unable to obtain information on additional patient-associated characteristics (e.g., socioeconomic status [eg., Education, occupation] and psychological factors [eg., reluctance to call family members for help or having no one around when experiencing symptoms to ask for help]) which may have impacted on patient’s medical care seeking behavior.\n\nOur data show that patients admitted for initial AMI in Hanoi, Vietnam experienced a significant delay time in seeking medical attention after the onset of AMI suggestive symptoms. Additional full-scale data of hospitalized patients with AMI across Hanoi will be needed in order to confirm these initial findings. Public educational programs for the general population, specifically targeting towards women, is greatly needed to incentivize patients to seek medical assistance immediately to ensure maximum effectiveness of treatments. Further research is required to understand potential factors associated with the prolonged delay in Vietnamese patients hospitalized with AMI and the most effective ways to encourage patients to seek medical care in a timely manner.\n\n\nData availability\n\nF1000Research: Dataset 1. Raw dataset for Nguyen et al., 2015 'Pre-hospital delay in Vietnamese patients hospitalized with a first acute myocardial Infarction: A short report', 10.5256/f1000research.6943.d10089223", "appendix": "Author contributions\n\n\n\nHLN, RJG conceived and designed the study. HLN, DTP, DAH, QNN carried out the study. HLN, RJG analyzed the data and prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nThe authors have declared that no competing interests exist.\n\n\nGrant information\n\nThis study was partially funded by the Global Health Office, University of Massachusetts Medical School, Worcester, MA, USA (Drs. Goldberg, Nguyen, and Ha). Additional support was provided by internal funding.\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank the doctors and nurses at the Vietnam National Heart Institute for their support and help.\n\n\nReferences\n\nAn international randomized trial comparing four thrombolytic strategies for acute myocardial infarction. The GUSTO investigators. N Engl J Med. 1993; 329(10): 673–82. PubMed Abstract \| Publisher Full Text\n\nNewby LK, Rutsch WR, Califf RM, et al.: Time from symptom onset to treatment and outcomes after thrombolytic therapy. GUSTO-1 Investigators. J Am Coll Cardiol. 1996; 27(7): 1646–55. PubMed Abstract \| Publisher Full Text\n\nNguyen HL, Ha DA, Phan DT, et al.: Sex differences in clinical characteristics, hospital management practices, and in-hospital outcomes in patients hospitalized in a Vietnamese hospital with a first acute myocardial infarction. PLoS One. 2014; 9(4): e95631. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNguyen HL, Nguyen QN, Ha DA, et al.: Prevalence of comorbidities and their impact on hospital management and short-term outcomes in Vietnamese patients hospitalized with a first acute myocardial infarction. PLoS One. 2014; 9(10): e108998. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTunstall-Pedoe H, Kuulasmaa K, Mähönen M, et al.: Contribution of trends in survival and coronary-event rates to changes in coronary heart disease mortality: 10-year results from 37 WHO MONICA project populations. Monitoring trends and determinants in cardiovascular disease. Lancet. 1999; 353(9164): 1547–57. PubMed Abstract \| Publisher Full Text\n\nMcManus DD, Gore J, Yarzebski J, et al.: Recent trends in the incidence, treatment, and outcomes of patients with STEMI and NSTEMI. Am J Med. 2011; 124(1): 40–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoldberg RJ, Steg PG, Sadiq I, et al.: Extent of, and factors associated with, delay to hospital presentation in patients with acute coronary disease (the GRACE registry). Am J Cardiol. 2002; 89(7): 791–6. PubMed Abstract \| Publisher Full Text\n\nGoldberg RJ, Yarzebski J, Lessard D, et al.: Decade-long trends and factors associated with time to hospital presentation in patients with acute myocardial infarction: the Worcester Heart Attack study. Arch Intern Med. 2000; 160(21): 3217–23. PubMed Abstract \| Publisher Full Text\n\nWang X, Hsu LL: Treatment-seeking delays in patients with acute myocardial infarction and use of the emergency medical service. J Int Med Res. 2013; 41(1): 231–8. PubMed Abstract \| Publisher Full Text\n\nSheifer SE, Rathore SS, Gersh BJ, et al.: Time to presentation with acute myocardial infarction in the elderly: associations with race, sex, and socioeconomic characteristics. Circulation. 2000; 102(14): 1651–6. PubMed Abstract \| Publisher Full Text\n\nNguyen HL, Gore JM, Saczynski JS, et al.: Age and sex differences and 20-year trends (1986 to 2005) in prehospital delay in patients hospitalized with acute myocardial infarction. Circ Cardiovasc Qual Outcomes. 2010; 3(6): 590–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQian L, Ji KT, Nan JL, et al.: Factors associated with decision time for patients with ST-segment elevation acute myocardial infarction. J Zhejiang Univ Sci B. 2013; 14(8): 754–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarshidi H, Rahimi S, Abdi A, et al.: Factors Associated With Pre-hospital Delay in Patients With Acute Myocardial Infarction. Iran Red Crescent Med J. 2013; 15(4): 312–6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan MS, Jafary FH, Faruqui AM, et al.: High prevalence of lack of knowledge of symptoms of acute myocardial infarction in Pakistan and its contribution to delayed presentation to the hospital. BMC Public Health. 2007; 7: 284. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerkins-Porras L, Whitehead DL, Strike PC, et al.: Pre-hospital delay in patients with acute coronary syndrome: factors associated with patient decision time and home-to-hospital delay. Eur J Cardiovasc Nurs. 2009; 8(1): 26–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaczynski JS, Yarzebski J, Lessard D, et al.: Trends in prehospital delay in patients with acute myocardial infarction (from the Worcester Heart Attack Study). Am J Cardiol. 2008; 102(12): 1589–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBanks AD, Dracup K: Factors associated with prolonged prehospital delay of African Americans with acute myocardial infarction. Am J Crit Care. 2006; 15(2): 149–57. PubMed Abstract\n\nNovak K, Aljinovic J, Kostic S, et al.: Pain to hospital times after myocardial infarction in patients from Dalmatian mainland and islands, southern Croatia. Croat Med J. 2010; 51(5): 423–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEl-Menyar A, Zubaid M, Rashed W, et al.: Comparison of men and women with acute coronary syndrome in six Middle Eastern countries. Am J Cardiol. 2009; 104(8): 1018–22. PubMed Abstract \| Publisher Full Text\n\nGoldberg RJ, Spencer FA, Fox KA, et al.: Prehospital Delay in Patients With Acute Coronary Syndromes (from the Global Registry of Acute Coronary Events [GRACE]). Am J Cardiol. 2009; 103(5): 598–603. PubMed Abstract \| Publisher Full Text\n\nNguyen HL, Saczynski JS, Gore JM, et al.: Age and sex differences in duration of prehospital delay in patients with acute myocardial infarction: a systematic review. Circ Cardiovasc Qual Outcomes. 2010; 3(1): 82–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcGinn AP, Rosamond WD, Goff DC Jr, et al.: Trends in prehospital delay time and use of emergency medical services for acute myocardial infarction: experience in 4 US communities from 1987–2000. Am Heart J. 2005; 150(3): 392–400. PubMed Abstract \| Publisher Full Text\n\nNguyen HL, Phan D, Ha D, et al.: Dataset 1 in: Pre-hospital Delay in Vietnamese Patients Hospitalized with a First Acute Myocardial Infarction: A Short Report. F1000Research. 2015. Data Source" }	[ { "id": "10208", "date": "13 Oct 2015", "name": "Felix Alvaro Medina Palomino", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is not clear how the author obtained their samples. There is a very small population to study and to obtain valid conclusions from. It is uncertain how the time of chest pain was recorded. It appears there is no system that allows for a correct determination of the precise moment the patient arrives at the ER, due to the fact that in many patients this particular data is missing. The report is based on more than a hundred patients but in fact the author only presents data from 65 patients. There is no explanation about the findings nor factors. Because only a few patients were enrolled, it is not possible to determine factors related to early or delayed arrival at the hospital. Finally some data, which are in fact results, are written in the methods.", "responses": [ { "c_id": "2910", "date": "01 Aug 2017", "name": "Hoa L. Nguyen", "role": "Author Response", "response": "It is not clear how the author obtained their samples. Response: We have described the study population in greater detail in the Case Ascertainment section of the Methods (page 3, para 2). In brief, lists of patients discharged from the Vietnam National Heart Institute in 2010 with possible AMI were obtained and International Classification of Disease-10 codes for possible AMI (I20-I25) were reviewed. The diagnosis of AMI was made on the basis of criteria developed by the World Health Organization which includes a suggestive clinical history, serum enzyme elevations above the hospital’s normal range, and serial electrocardiographic (ECG) findings during hospitalization consistent with the presence of AMI; at least 2 of these 3 criteria needed to be present for an AMI to have occurred. We restricted our patient population to those living in the city of Hanoi and with information on prehospital delay recorded in hospital medical records. We have added this information in the revised manuscript. 2. There is a very small population to study and to obtain valid conclusions from.Response: We have acknowledged this limitation in the revised discussion. In our conclusions, we have emphasized the need for full-scale surveillance of Hanoi residents hospitalized with AMI at all Hanoi hospitals to confirm these preliminary descriptive findings. 3. It is uncertain how the time of chest pain was recorded. It appears there is no system that allows for a correct determination of the precise moment the patient arrives at the ER, due to the fact that in many patients this particular data is missing.Response: Due to the design of this retrospective study, whose information was based exclusively on information contained in hospital medical records, there was no systematic method to collect and record the time of onset of symptoms suggestive of AMI (e.g., chest pain) since these data were collected and recorded in hospital records by nurses and physicians with varying degrees of expertise and interest in collecting this information. Data on duration of prehospital delay were missing in approximately two thirds of Hanoi residents hospitalized with AMI in 2010, which was mainly due to missing data on the time of onset of acute symptoms suggestive of AMI, and not ER arrival time. We have included this limitation in the discussion section of the revised manuscript. 4. The report is based on more than a hundred patients but in fact the author only presents data from 65 patients.Response: The present report is based on 103 patients with independently confirmed AMI. Data on duration of prehospital delay (continuous outcome) were available in only 65 patients, whereas the other 38 patients had data available only for a time interval (categorical outcome). Inasmuch, we have analyzed duration of prehospital delay as a continuous outcome in 65 patients and as a categorical outcome in 103 patients. We have modified the results section to reflect our approaches to the analysis of these data in the revised manuscript. 5. There is no explanation about the findings nor factors. Response: In this brief report, our primary goal was to present descriptive data about the extent of prehospital delay in patients hospitalized with AMI at the major teaching hospital in Hanoi, information which is unknown and sorely lacking in Vietnam. Since the sample size is small, we were unable to examine factors associated with prolonged delay, information which is important for designing and implementing various educational intervention approaches to improve patient’s care seeking behavior. We have emphasized this limitation and the need for future studies in the revised manuscript. 6. Because only a few patients were enrolled, it is not possible to determine factors related to early or delayed arrival at the hospital.Response: Please see the above response (#5). 7. Finally some data, which are in fact results, are written in the methods.Response: We have removed data on prehospital delay in the method sections and added them into the results section of the revised manuscript." } ] }, { "id": "15408", "date": "03 Aug 2016", "name": "Dao Thi Mihn An", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThere were not any official publications in Vietnam regarding issue of pre-hospital delay in Vietnamese patients hospitalized with a first acute myocardial infarction until now. Therefore, the short report on this issue written by Hoa et al is highly appreciated.\nThis study was conducted with a small and convenient sample of 103 AMI patients with their first AMI and the study approach was to review AMI patients' medical records after their discharge that must be embedded biases as it was a retrospective study. The inconsistence of asking and identifying time of delay by different health workers at the time of hospitalization and the different subjects who supplied information for health workers should be considered the main problems of the validity of data in this study beside 3 other limitations that the author has discussed on this paper. Moreover, it was likely that there were missing value regarding delay time occurred during process of extracting data from AMI patients' medical record.\nHowever, this study initially indicated 2 very important findings that pre-hospital delay time for hospitalization among AMI patients was much longer than that of AMI patients in the local and worldwide and patients who had a delay time over 6 hours were more likely to be female compared to -their counterparts. These findings make help in giving suggestion for further full-scale data of hospitalized patients with AMI across Hanoi in order to confirm these initial findings.\n\nI would like very much to approve for this short report as it is the starting point for further large-scale study in this issue in Vietnam where such information are still rare.", "responses": [ { "c_id": "2911", "date": "01 Aug 2017", "name": "Hoa L. Nguyen", "role": "Author Response", "response": "Response: Due to the retrospective nature of this observational study, there was no systematic method established to record the time of onset of symptoms suggestive of AMI (e.g., chest pain) in a standardized manner by trained health professionals. As noted in response to a similar query by the other reviewer, this information was abstracted from notes written by physicians and nurses and emergency personnel which may have been collected and recorded in a non-standardized manner. Duration of prehospital delay was missing in approximately two thirds of Hanoi residents hospitalized with AMI in 2010, which was mainly due to missing data on the time of onset of symptoms suggestive of AMI. We have noted this limitation in the discussion section of the revised manuscript." } ] } ]	1	https://f1000research.com/articles/4-633
https://f1000research.com/articles/4-214/v1	10 Jul 15	{ "type": "Research Article", "title": "Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model", "authors": [ "Mark Brenneman", "Amanda Field", "Jiandong Yang", "Gretchen Williams", "Leslie Doros", "Christopher Rossi", "Kris Ann Schultz", "Avi Rosenberg", "Jennifer Ivanovich", "Joyce Turner", "Heather Gordish-Dressman", "Douglas Stewart", "Weiying Yu", "Anne Harris", "Peter Schoettler", "Paul Goodfellow", "Louis Dehner", "Yoav Messinger", "D. Ashley Hill", "Mark Brenneman", "Amanda Field", "Jiandong Yang", "Gretchen Williams", "Leslie Doros", "Christopher Rossi", "Kris Ann Schultz", "Avi Rosenberg", "Jennifer Ivanovich", "Joyce Turner", "Heather Gordish-Dressman", "Douglas Stewart", "Weiying Yu", "Anne Harris", "Peter Schoettler", "Paul Goodfellow", "Louis Dehner", "Yoav Messinger" ], "abstract": "Pleuropulmonary blastoma (PPB) is the most frequent pediatric lung tumor and often the first indication of a pleiotropic cancer predisposition, DICER1 syndrome, comprising a range of other individually rare, benign and malignant tumors of childhood and early adulthood. The genetics of DICER1-associated tumorigenesis are unusual in that tumors typically bear neomorphic missense mutations at one of five specific “hotspot” codons within the RNase IIIb domain of DICER 1, combined with complete loss of function (LOF) in the other allele. We analyzed a cohort of 124 PPB children for predisposing DICER1 mutations and sought correlations with clinical phenotypes. Over 70% have inherited or de novo germline LOF mutations, most of which truncate the DICER1 open reading frame. We identified a minority of patients who have no germline mutation, but are instead mosaic for predisposing DICER1 mutations. Mosaicism for RNase IIIb domain hotspot mutations defines a special category of DICER1 syndrome patients, clinically distinguished from those with germline or mosaic LOF mutations by earlier onsets and numerous discrete foci of neoplastic disease involving multiple syndromic organ sites. A final category of patients lack predisposing germline or mosaic mutations and have disease limited to a single PPB tumor bearing tumor-specific RNase IIIb and LOF mutations. We propose that acquisition of a neomorphic RNase IIIb domain mutation is the rate limiting event in DICER1-associated tumorigenesis, and that distinct clinical phenotypes associated with mutational categories reflect the temporal order in which LOF and RNase IIIb domain mutations are acquired during development.", "keywords": [ "DICER1 truncation", "PPB", "Pleuropulmonary blastoma", "Mosaicism", "Paediatric cancer", "RNAse IIIb" ], "content": "Introduction\n\nPleuropulmonary blastoma (PPB) is the most common primary lung cancer of childhood (OMIM #601200)1,2. Early PPB (type I) presents as lung cysts that are at risk for transformation into high grade sarcomas, which may have both cystic and solid components (PPB type II) or be entirely solid (PPB type III)2,3. Not all PPB type I cysts progress to sarcoma; those that do not are designated type Ir (regressed)1,3. The genetic and epigenetic events responsible for initiation of cyst formation and subsequent progression to sarcoma are just beginning to be understood3–6. PPB is pathognomonic for a childhood cancer syndrome that features a range of other benign and malignant neoplasms including ovarian Sertoli-Leydig cell tumor (SLCT), cystic nephroma (CN) and renal sarcoma or Wilms tumor, nodular hyperplasia and carcinoma of the thyroid gland, nasal chondromesenchymal hamartoma (NCMH), embryonal rhabdomyosarcoma (ERMS), pituitary blastoma and pineoblastoma2,4,7–30. We previously identified inherited loss of function (LOF) mutations in DICER1 (OMIM #606241) as the major genetic factor in this syndrome4. DICER1 syndrome thus became the first cancer predisposition associated with a systemic defect in microRNA (miRNA) processing.\n\nThe DICER1 gene encodes an RNase III-family endonuclease that cleaves precursor microRNAs (pre-miRNA) into active miRNA31,32. Sequencing studies of syndromic tumors have revealed biallelic, compound mutations of DICER16,11,15,21,26,28–30,33–35. Generally, one allele (often germline) bears a nonsense or frame-shift mutation predicted to cause full loss of function (LOF), and one allele bears a missense mutation in the DICER1 RNase IIIb domain. Biallelic LOF mutations have not been identified in PPB, suggesting that retention of some miRNA processing function is usually required for tumor survival6,35. RNase IIIb missense mutations in DICER1 syndrome tumors affect five \"hotspot\" codons that encode key amino acids in the metal-binding catalytic cleft of the nuclease domain: E1705, D1709, G1809, D1810 and E18136,26,29,30,33–35. Amino acid substitutions at these positions cause neomorphic DICER1 function in miRNA processing, such that cleavage of mature 5p miRNAs from the 5’ end of pre-miRNA hairpin structures fails, while mature 3p miRNAs continue to be cleaved from the 3’ end normally6,26,33,35,36. The high overall ratio of 5p to 3p mature miRNAs seen in normal tissues is essentially inverted in DICER1 tumors, suggesting that uncleaved 5p miRNAs are rapidly degraded6. Depletion of 5p miRNAs alters expression of numerous downstream target mRNAs across the exome, including some critical for embryogenesis or tumor suppression33,36. The pleiotropic nature of DICER1 syndromic disease likely reflects the diverse array of genes regulated by miRNAs during organ development and in differentiated tissues.\n\nClinical features of DICER1 syndrome are highly variable with regard to age at first occurrence of neoplastic disease, the number of discrete foci of disease that develop over time, and the specific organ sites involved. As a step toward understanding the basis of clinical variability, we explored the spectrum of predisposing DICER1 mutations in a large cohort of PPB/DICER1 syndrome patients. Correlation of genotypes with clinical features revealed a distinctive phenotype of early onsets and extensive, multifocal disease in patients who are mosaic for hotspot missense mutations in the RNase IIIb domain. We propose that the extreme phenotypes of this patient group are attributable to the order in which allelic DICER1 mutations were acquired during development, i.e., an RNase IIIb hotspot missense mutation acquired early in embryogenesis and subsequently unmasked by LOF mutations or loss of the second allele. Understanding how the interplay of RNase IIIb missense and LOF mutations influences the expression of syndromic neoplasias can aid diagnosis at early stages, when they are most curable, and improve genetic evaluation and counseling for families with DICER1 syndrome.\n\n\nSubjects and methods\n\nPPB patients (n = 124) and family members were ascertained through the International PPB Registry (IPPBR). Inclusion into this study required a pathologic diagnosis of PPB verified by central review (LPD, DAH). All subjects gave written consent for molecular and family history studies, as approved by the Human Research Protection Offices at Washington University in St. Louis (HSC#04-1154), Children's Hospitals and Clinics of Minnesota (IRB#98107), and Children's National Medical Center (IRB#4603; Pro0315). For families with more than one affected member, only data from the initial proband is included. Medical history and biological samples were collected and prepared for analysis as previously described4,30. Tumor tissue was available for sequencing from a subset of patients. For two of these cases, DNA was isolated from unstained tissue on glass slides using the Pinpoint Slide DNA Isolation System (Zymo, Irvine, CA).\n\nClinical data was abstracted from medical records and imaging studies. All children had pathologic confirmation of PPB. In addition, lung cysts, kidney cysts, CN, NCMH, SLCT, ERMS, thyroid cancer or nodules, pineoblastoma and/or pituitary blastoma were defined as evidence of syndromic disease. Lung cysts that were distinctly separate (anatomically separate in same lobe or in different lobes) were counted individually.\n\nInitial sequencing of blood and saliva DNA samples was by standard Sanger methods described previously4 or by a commercial laboratory (Ambry Genetics, Aliso Viejo, CA). Low-frequency variants were detected and quantified by targeted next-generation sequencing (NGS) using a custom multiplex PCR panel for DICER1 coding regions (Ion Torrent Ampliseq, Life Technologies, Grand Island, NY, USA) (Table S1)30. NGS was performed on an Ion Torrent 318 v2 chip (ION PGM Sequencing 200 kit v2, Life Technologies) with an average of 6 samples per chip, to achieve an average depth of coverage of 3000 filtered reads. Signal processing, mapping and quality control were performed with Torrent Suite software v.4.0.2 (Life Technologies). Variant calls were made using the Torrent Variant Caller Plugin v.4.0, with somatic low stringency mutation workflow and default settings. BAM files of raw reads were reviewed using Integrative Genomics Viewer v2.337,38.\n\nDICER1 sequence variants were annotated with Alamut Batch software (Interactive Biosoftware, Rouen, France), with reference to DICER1 transcript record NM_177438.2. Nonsense, frameshift and canonical splice-site mutations were considered loss of function (LOF). Missense variants affecting codons 1705, 1709, 1809, 1810 and 1813 in the RNase IIIb domain were classified as “hotspot” mutations. For variants assayed by NGS, allele frequencies were calculated from filtered read counts. The SIFT algorithm was used to assess potential significance of novel missense mutations39–41. All variants identified were deposited into ClinVar (accession numbers SCV000195560-SCV000195643). The numbers of possible single-nucleotide changes that can produce amino acid substitutions at the five hotspot codons or nonsense mutations anywhere in the DICER1 open reading frame, or disrupt canonical splice sites, were compiled from DICER1 transcript record NM_177438.2 and genomic record NG_016311.1.\n\nMolecular probes for NanoString Copy Number Assay at the DICER1 locus were developed in collaboration with NanoString Technologies, Inc., Seattle, WA (Table S2). Genomic DNA was fragmented and hybridized using the nCounter Prep Station, and hybridization signals quantified using the nCounter Digital Analyzer, according to NanoString’s recommendations. Preliminary analysis and quality control of the data were performed using nSolver Analysis Software version 1.1 (NanoString) with default copy number variation (CNV) analysis settings. CNVs were confirmed with high-density CNV array hybridization in a commercial laboratory (Prevention Genetics, Marshfield, WI).\n\nThe number of disease foci per patient and the age at DICER1 syndrome diagnosis were compared between mutation categories using nonparametric tests, due to the skewness of both clinical features and to the unbalanced sample sizes. Kruskal-Wallis tests were used to compare medians among the four mutation categories. Where a significant overall association was found, pair-wise post-hoc Wilcoxon rank sum tests were used to compare medians, and resulting p-values adjusted for multiple comparisons using the Sidak method. A p-value of 0.05 was considered statically significant and all analyses were performed using Stata V13 (College Station, TX).\n\n\nResults\n\nOur overall approach to detecting and categorizing predisposing DICER1 mutations in PPB children is shown schematically in Figure 1. We identified germline, heterozygous DICER1 mutations in 90 of the 124 probands in our cohort (72.6%; Table 1, Table S3). Nearly all (89) were detected by Sanger sequencing of exonic PCR amplicons. For one child in whom no mutation was detected by Sanger sequencing, blood DNA was probed by NanoString hybridization, which indicated deletion of one copy of exon 24. High-density CNV array hybridization was used to confirm a heterozygous deletion of ~ 1.1 kb, comprising all of exon 24 and parts of the flanking introns (c.5096-498_5364+356del). Paternal DNA was positive for the deletion, which was anticipated as this child has an uncle with CN. Only one previous instance of a large, intragenic deletion as a germline DICER1 mutation has been reported, which suggests such mutations are very rare42. The actual prevalence of large deletions is difficult to estimate because they are not readily detected by the targeted sequencing strategies applied for mutation screening in this study and most others.\n\nAbbreviations: PPB pleuropulmonary blastoma; NCMH nasal chondromesenchymal hamartoma; ERMS embryonal rhabdomyosarcoma.\n\na. Age at first clinical presentation with PPB or other DICER1 syndrome pathology.\n\nb. Total number of discrete disease foci, as defined in Subjects and Methods.\n\nc. One patient with both type IR and type II PPB.\n\nd. Medians compared using a Kruskal-Wallis test; post-hoc pair-wise tests adjusted for multiple comparisons.\n\nA cohort of 124 children diagnosed with pleuropulmonary blastoma (PPB) was screened for predisposing DICER1 mutations by targeted Sanger sequencing and/or low-depth, next-generation sequencing (NGS) of DNA amplified from peripheral blood cells, saliva (buccal cells) or non-neoplastic surgical specimens. Sequenced PCR amplicons covered the 26 coding exons of the DICER1 open reading frame and flanking splice signals. DICER1 coding sequence or splice site mutations detected at approximately heterozygous frequency in blood or normal tissue cells were categorized as germline mutations. For patients in whom screening revealed no germline mutation, blood and/or normal tissues were analyzed for the presence of intragenic deletions or larger genomic alterations using NanoString copy number assay and CNV array, and for coding or splice site mutations present at low allele frequencies using high-depth NGS on the Ion Torrent platform. Wherever possible, matched tumor specimens were also sequenced on the Ion Torrent platform. Low-frequency DICER1 mutations detected in multiple normal and/or tumor samples, or in primary tumors of multiple organs, were categorized as mosaic mutations. Mosaic mutations were further categorized as loss-of-function (LOF) or RNase IIIb domain hotspot missense mutations. Patients for whom both LOF and hotspot mutations were identified in a single tumor, but not found in blood or normal tissue samples, were categorized as having tumor-specific, biallelic DICER1 mutations.\n\nThe spectrum of germline mutations is dominated by truncating, LOF mutations (Figure 2). These are mainly single-nucleotide substitutions that produce new stop codons (33 cases, 37%) and small insertions or deletions (indels) within exons that shift reading frame (44 cases, 49%). Seven mutations of consensus splice sites occur in our cohort; of which six are predicted to cause exon skipping during transcript splicing with resulting frameshift. The remaining splice site mutation, c.1752+1delG, is at the 5’ end of intron 10. Skipping of exon 10 would cause in-frame deletion of 81 amino acids near the end of the helicase domain. In all, 84 of 90 germline DICER1 mutations discovered in patients (93%) truncate the open reading frame before the end of the critical RNase IIIb domain, and are thus predicted to result in complete loss of DICER1 protein function even if the message escapes nonsense-mediated decay. Six non-truncating germline mutations were identified, including the intron 10 splice site mutation described above and five non-hotspot missense changes: I582T, L1583R and G1708E (each seen once) and D1822V (identified in two patients) (Table S4). The I582T substitution is at the distal end of the helicase domain (Figure 2), the role of which is unclear. L1583R is within the RNase IIIa domain and segregates with disease in a family4. The G1708E and D1822V mutations both fall within the RNase IIIb domain, near the metal-binding catalytic site. These two missense mutations are predicted to compromise protein function by the SIFT algorithm but their precise functional significance in DICER1 is unknown39–41.\n\nA linear schematic of the DICER1 open reading frame is shown with annotated functional domains represented to scale. Sequence changes identified as inherited or de novo germline mutations in 90 PPB/DICER1 syndrome patients are indicated by position along the coding sequence. Mutations linked to the schematic by two, three or four fine lines are those discovered in a corresponding number of individuals from unique families.\n\nDNA was available from both parents for 77 children with germline mutations, and Sanger sequencing of parental DNA was sufficient to confirm 67 of the mutations (87%) as inherited. Mutations in the ten patients whose parents had no DICER1 mutation detected by Sanger sequencing were provisionally considered de novo. To confirm this, targeted next generation sequencing (NGS) was performed in eight of the ten trios, yielding mutant allele frequencies between 42.0% and 57.1% in the probands but no conclusive evidence of the variants in parental blood. There were no statistically significant differences between de-novo and inherited germline LOF patients with respect to age at onset, numbers of disease foci or survival.\n\nPenetrance of familial DICER1 LOF mutations was far from complete. Of the 67 families in this cohort with segregating LOF mutations, 29 include parents or siblings who are confirmed as mutation carriers but have no history of syndromic disease (Table S5). True penetrance is difficult to estimate because we have limited knowledge of how many germline DICER1 mutation carriers are phenotypically normal, as only a subset with overtly affected family members have been ascertained. Moreover, subclinical disease is common. Preliminary data from an ongoing NCI-sponsored DICER1 family history study indicate that ~ 87% of otherwise asymptomatic individuals with confirmed DICER1 mutations have thyroid nodules detectable by ultrasound and ~ 43% have lung cysts detectable by CT scan (D.R. Stewart and L. Doros, unpublished).\n\nAmong children with germline LOF mutations, age at first diagnosis of PPB or other syndromic disease was typically one to five years (70 of 90 patients), but this ranged from diagnosis within days of birth to as late as eighteen years. The most frequent syndromic condition after PPB was cystic nephroma, followed by thyroid disease (nodular hyperplasia or carcinoma), nasal chondromesenchymal hamartomas and embryonal rhabdomyosarcomas (Table 1, Table S5). The number of discrete disease foci per patient ranged as high as five or six (in two patients), but the majority of children in this group had experienced no more than two at the time of their most recent exam, and nearly half had only a single PPB tumor. None of the six patients with non-truncating germline mutations had unusual clinical features and as a group they were not distinguishable from patients with truncating mutations. Table S6 provides data on somatic hotspot mutations identified in all available tumors of PPB children.\n\nWe and others have previously described biallelic DICER1 mutations in tumors of children who apparently have no germline mutation, inherited or de novo6,14,35. Because PPB children are typically so young when affected, we hypothesized that at least some cases of this kind reflect mosaicism, i.e., a mutation present in some but not all cells of the body, because it occurred during post-zygotic embryonic development rather than being present in the zygote (as a germline mutation would be). To explore this possibility, we performed targeted, high-depth NGS of DICER1 coding exons in DNA from blood and/or other normal tissues of children who had tested negative for germline mutation by Sanger sequencing, and in matched samples of tumor tissue where available. We categorized a DICER1 mutation detected by NGS as mosaic when the following criteria were met: i. The mutation was evidently not a constitutional, germline allele because it was present at sub-heterozygous frequency (arbitrarily taken as below 35% of reads) in peripheral blood and/or other normal tissue samples. ii. The mutation was evidently not specific to a tumor, because the same mutant allele was detected in one or more normal, non-neoplastic tissue samples, OR, the same mutant allele was detected in multiple primary tumors arising in different organs (Figure 1). We identified ten children with predisposing mosaicism for either LOF or RNase IIIb hotspot mutations (Table 1).\n\nMosaic LOF mutations were detected in five children, at frequencies that ranged from 1.1% to 17.2% of allelic reads in DNA from blood, saliva or normal fibroblasts (Table S7). For three of these children, archival PPB tumor tissue was available, and in each the LOF mutation was present, as was an RNase IIIb domain hotspot mutation. Two of the five children with mosaic LOF mutations had a single focus of disease in a lung. The other three children each had two foci of disease, also restricted to the lungs. It might be anticipated that children bearing mosaic LOF mutations tend to have fewer disease foci than those with germline LOF mutations because the number of cells at risk for second hits is generally lower. No statistically significant difference of this kind can be discerned from the five mosaic LOF children in our cohort, but notably, none have developed syndromic tumors other than PPB. As this was not a population study, we cannot estimate how many persons with mosaic LOF mutations are asymptomatic but, by analogy to the low penetrance of familial LOF mutations, it could be a large proportion.\n\nFive children harbored mosaic RNase IIIb domain hotspot missense mutations, detected in multiple primary neoplasms and/or non-neoplastic tissues (Table 2). None had family members with features of DICER1 syndrome, and the RNase IIIb hotspot mutations found in probands were not detected in parental blood, consistent with a postzygotic origin. NGS of tumor tissues from these children identified somatic LOF mutations or allele loss in some but not all specimens, with the caveat that allele loss can be difficult to detect in specimens with low tumor purity (i.e., PPB Type Ir, CN and NCMH). For one mosaic hotspot patient, specimens of a thyroid carcinoma and two separate ovarian Sertoli-Leydig cell tumors (SLCT) were available for NGS. The thyroid carcinoma and one SLCT had lost the second DICER1 allele, but the other SLCT had instead sustained a frameshift mutation (Table 2). This is consistent with underlying mosaicism for the RNase IIIb hotspot mutation and subsequent acquisition of independent LOF mutations in each tumor site.\n\nAbbreviations: Ca carcinoma; CN cystic nephroma; LOF loss of function; NCMH nasal chondromesenchymal hamartoma; ND none detected; NR not reported; PPB pleuropulmonary blastoma; SLCT Sertoli-Leydig cell tumor.\n\na. Percent tumor cells in specimen, estimated visually by microscopy in tumor sections.\n\nb. Allele frequency estimates were derived from NGS read counts in this study. In the two cases reported by Klein et al, allele frequencies were determined by pyrosequencing assays.\n\nc. Hotspot allele detected by Sanger sequencing only; no NGS performed.\n\nd. Variant allele frequency below estimated error rate for base substitutions (0.07%) with Ion Torrent using 200 bp sequencing kit50.\n\nThe five children with mosaic RNase IIIb domain hotspot mutations shared unusual clinical features. All were diagnosed with DICER1 syndrome early; within 15 months of birth. All presented with multiple cysts of the lungs and/or kidneys, which were accompanied or followed in all cases by multiple DICER1 syndromic tumors (Figure 3). Four of the five had CN as well as PPB. Other tumors included SLCT, thyroid nodular hyperplasia or carcinoma, NCMH, ciliary body medulloepithelioma and pineoblastoma. In addition, four of the five children experienced episodes of intestinal intussusception, which in three cases were associated with polyps of the small intestine discovered upon surgical intervention. Total numbers of discrete disease foci per patient were extraordinarily high, ranging from a minimum of 10 to as many as 24. Despite the small number of patients in this group, statistical analysis confirms clinical impressions that they are distinct from those with predisposing LOF mutations. Mean age at first DICER1 syndrome diagnosis was significantly earlier, and both mean and median numbers of disease foci are significantly greater in children with mosaic RNase IIIb mutations (Table 1). The association with juvenile-type intestinal polyps and intussusception is a novel feature of children with mosaic RNase IIIb hotspot mutations, not previously seen in children with other categories of DICER1 mutation.\n\nFor each of the five mosaic hotspot children identified in this study, an individual timeline indicates numbers of discrete foci of neoplastic disease and their histopathological types, graphed with respect to patient age at diagnosis. Across the lower portion of the chart, a single aggregate timeline (dark violet) represents the mean number of disease foci for all PPB/DICER1 syndrome patients with predisposing loss of function (LOF) mutations identified in this study, graphed with respect to patient age at diagnosis. The shaded areas (in lighter violet) surrounding the timeline for LOF mutation patients indicates one and two standard deviations above and below the mean. The range of foci number among all LOF mutation patients was 0 to 6 in all years of age represented (not shown). Abbreviations: CN cystic nephroma; CBME ciliary body medulloepithelioma (eye); NCMH nasal chondromesenchymal hamartoma; PPB pleuropulmonary blastoma, PinB pineoblastoma; SIP small intestinal polyp(s); SLCT Sertoli-Leydig cell tumor (ovary); TCa thyroid carcinoma; TN thyroid nodule(s).\n\nFour of the five children with mosaic DICER1 hotspot mutations presented with cystic PPB (type I/IR) rather than sarcomatous disease (type II or type III) and all five have survived to date. However, this does not indicate a benign clinical course. Though all five hotspot mosaic children are alive, their clinical experiences have been complicated and arduous (Figure 3). Each has undergone multiple major surgeries and chemotherapies with concomitant morbidities. One child is alive with recurrent disease at last follow-up.\n\nIn twelve children, we identified biallelic DICER1 mutations present at high allele frequencies in a PPB tumor, but not detectable in blood even with the benefit of high-depth NGS (Table S8). Tumors from these children had an RNase IIIb hotspot missense mutation and either a nonsense LOF mutation (n = 5) or allele loss (n = 7). All twelve children presented with a single PPB tumor and none developed additional foci of disease in the lungs or other organs over the course of subsequent follow-up. This is consistent with occurrence of both an RNase IIIb hotspot mutation and a LOF mutation or allele loss within a single, highly localized clone of somatic cells which then gave rise to the tumor. Absence of additional disease foci is a predictable outcome if both DICER1 mutations are restricted to the initial site of tumorigenesis. However, the absence of additional disease foci among children in this category did not indicate less dangerous disease. Of the 12 patients, 11 had advanced PPB (type II or III), and two succumbed (Table 1).\n\nTwelve PPB probands in our cohort are negative for predisposing DICER1 mutations detectable in blood DNA by Sanger sequencing or NGS of coding exons. Ten of these children had a single focus of disease, and thus may be sporadic cases involving tumor-specific, biallelic DICER1 mutations, but tumor tissue is either not available or not of sufficient quality for confirmation. Two had multifocal disease, possibly reflecting DICER1 mosaicism that is not well represented in the blood lineage (below our limits of detection). Clinical features of the twelve unresolved cases and the status of further analyses pending or completed, including tumor sequencing, NanoString copy number assay and germline sequencing for additional candidate loci, are summarized in Table S9.\n\n\nDiscussion\n\nAll germline DICER1 truncating mutations are predicted to be essentially equivalent in their effect: complete loss of function in miRNA processing. This prediction is based partly on nonsense-mediated decay, but also reflects the functional domain structure of the DICER1 protein. All truncating mutations so far identified in PPB/DICER1 syndrome patients interrupt the open reading frame before the end of the critical RNase IIIb domain (Figure 1, Table S3). Neomorphic RNase IIIb domain function (skewed 5p/3p miRNA production) is a recurring feature of DICER1 tumors, and it is plausible that loss of all wildtype RNase IIIb function is required for it to become tumorigenic. Presumed equivalence of all truncating mutations is consistent with clinical findings: no correlations are apparent between locations of germline truncating mutations within the DICER1 gene and clinical features such as age of onset, number of disease foci, specific tissue sites involved or survival. Non-truncating germline mutations are too rare for correlations with clinical presentations or outcomes to be ascertained.\n\nThe natural history of PPB indicates a multistep genetic pathogenesis, and so it is not surprising that in some cases where no germline DICER1 mutation can be detected, one of the two “hits” required for tumorigenesis was acquired during embryogenesis in the form of somatic mosaicism. Mosaic mutations may ultimately prove important in the pathogenesis of many other sporadic childhood neoplasias, as demonstrated recently for retinoblastoma (RB1)43.\n\nMosaicism for RNase IIIb domain hotspot missense mutations defines a special category of DICER1 syndrome patients that are phenotypically distinct from those who bear germline or mosaic LOF mutations. RNAse IIIb hotspot mutations have not been encountered as inherited alleles in this study or others, which suggests they are inviable4,8,10,11,14–16,21,26–30,34,44. In addition to the five mosaic RNase IIIb hotspot patients in our cohort, three apparently similar cases have been reported (Table 2). Klein et al. described two infants with bilateral Wilms tumor and multiple cysts of the kidneys and lungs44. Each child was found to be mosaic for a DICER1 RNase IIIb domain missense mutation, although in one case the mutation was at D1713; also an acidic residue within the RNase IIIb catalytic cleft, but not an established hotspot. De Kock et al. described an infant with pituitary blastoma and bilateral cysts of the kidneys and lungs in whom a de-novo hotspot mutation was detected at high allele frequency in blood as well as tumor11.\n\nClinically, mosaic hotspot patients are distinguished by two features: i.) consistently early presentations of neoplastic disease, often by one year of age, and ii.) numerous discrete foci of disease developed concurrently or successively, usually involving more than one syndromic tissue/organ site (Figure 3, Table 2). The two features are related and can be interpreted within the conceptual framework of the emerging model for DICER1 syndrome pathogenesis, which provides important insight as to how tumor suppression by DICER1 fails6,26,33,35,36. DICER1 is not a classical tumor suppressor gene for which “two hits” – loss of function in both alleles – are required to allow tumorigenesis. Neither is it haploinsufficient in the usual sense, i.e., that cells with only one expressed allele make wild-type protein, but not in sufficient quantity to fulfill its function. Rather, it is neomorphic function by mutant DICER1 protein, with substitutions of key amino acids in the RNase IIIb domain that causes tumor suppression to falter when it is not masked by expression of wild-type DICER1 protein. Unmasking of an RNase IIIb hotspot mutation may arise through any form of LOF mutation in the wild type allele, including allele loss. The two mutational events, RNase IIIb missense and LOF, may occur in either order and both are generally required to foment the initiation of tumorigenesis. However, as outlined below, RNase IIIb hotspot mutation is a low-probability event and LOF mutation is, relatively, a very high-probability event. The projected consequence of these lopsided probabilities is that occurrence of an RNase IIIb hotspot mutation becomes the rate-limiting step in onset of pathogenesis.\n\nThe RNase IIIb domain hotspots in DICER1 are a diminutive mutational target; five codons within an open reading frame of 1922 codons (0.26%). Moreover, molecular mechanisms by which RNase IIIb hotspot missense mutations can arise are restricted to errors of DNA replication and/or DNA repair that produce nucleotide substitution without disturbing the open reading frame. There are 36 possible single-nucleotide changes that can produce amino acid substitutions at these five codons, and only a subset of them has ever been identified in DICER1 syndrome tumors. The spectrum of pathogenic RNase IIIb hotspot mutations is thus very narrow. In contrast, the spectrum of possible LOF mutations is broad and mechanistically diverse. Of the 1922 codons in the DICER1 open reading frame, 675 can be converted to a stop codon by a single nucleotide change. A subset can be converted in more than one way, giving a total of 736 possible single nucleotide changes that result in a nonsense mutation. Among the other 16,562 possible single nucleotide changes in the DICER1 open reading frame, presumably some would be missense mutations that disrupt DICER1 protein function. The five non-hotspot missense mutations we detected as predisposing alleles in PPB probands are likely examples (Figure 2). The individual nucleotides of the DICER1 open reading frame present 5766 point locations at which insertion or deletion of one or a few nucleotides can shift reading frame. An additional 104 bases comprise canonical splice sites of the 26 DICER1 introns, where small sequence changes may result in exon skipping, with or without frameshift. The possibilities for LOF mutations also include larger intra-locus deletions and allele loss through copy-neutral loss of heterozygosity, segmental deletions or complete loss of chromosome 14. Absolute frequencies of these diverse DICER1 mutational mechanisms in a particular cell lineage cannot be modeled precisely, but it becomes clear that the aggregate likelihood of all possible LOF mutations is vastly greater than the likelihood of a neomorphic mutation in one of the five hotspot codons.\n\nIt follows that in a developing embryo or child with a germline (or mosaic) DICER1 LOF mutation, “second hits” occurring in a somatic cell will almost always be another LOF mutation, usually resulting in cell death or limited proliferation at most. Rarely, a second hit will be an RNase IIIb hotspot missense mutation, which allows for continuing cell viability and growth, though at the cost of skewed miRNA processing that may ultimately promote tumorigenesis in the surviving clones of cells. However, the low likelihood of incurring an RNase IIIb hotspot missense mutation in somatic cells means that months, years or a lifetime may elapse before one occurs. Further, the developmental context in which a second, hotspot mutation occurs may be important. There are apparently windows of risk for transformation, perhaps coinciding with certain periods of organ/tissue development when an “onco-fetal” gene program is normally active and subject to miRNA modulation, i.e., lung, kidney and brain in the embryo; uterine cervix and ovaries in pubertal girls1–3,8,9,45. A low probability of RNase IIIb hotspot mutations as second hits during windows of risk may underlie the low penetrance and variable expression of familial LOF mutations in DICER1 syndrome.\n\nFor a developing child with a mosaic RNase IIIb hotspot mutation, the prospects are radically different. Somatic cells that bear the RNase IIIb hotspot mutation, masked by a wild type allele, will be viable and non-tumorigenic unless and until they sustain a second hit. However, cells with a preexisting RNase IIIb hotspot mutation are at high aggregate risk of acquiring a subsequent LOF mutation, because it can take any of the myriad forms outlined above. The probability of a secondary LOF mutation occurring during expansion of any given cell lineage over the course of prenatal and postnatal development is relatively high, and independent LOF mutations in multiple lineages may occur. If sufficient fractions of cells in critical lineages are affected, disturbed regulation of developmental gene expression programs arising from defective miRNA processing may be lethal in utero. For surviving embryos, onsets of tumorigenesis will tend to be early and, depending on embryonic distribution of the RNase IIIb hotspot mutation, foci of tumorigenesis may arise in one or more organ sites characteristic of DICER1 syndrome. Additionally, we hypothesize that in mosaic hotspot children, wider tissue/organ distribution of aberrant miRNA processing during development may produce syndromic features not seen in children with predisposing LOF mutations, such as juvenile-type small intestinal polyps, or the generalized somatic overgrowth noted by Klein et al44.\n\nRecent publications have outlined general recommendations for mutation detection and clinical evaluation for syndromic disease in patients with suspected DICER1 syndrome and family members9,46–48. Here we add considerations of risk for multifocal disease and reproductive transmission of DICER1 mutations based on mutation category.\n\nMost predisposing DICER1 mutations are germline and detectable by targeted Sanger sequencing from blood. Initial testing should include parents, to distinguish inherited from de novo mutations. Sanger sequencing will usually suffice to detect a parental mutation that is also constitutional, but may fail to detect mosaicism. There is growing appreciation that apparently de novo mutations in children with genetic disease sometimes stem from mosaicism in a parent, which can often be detected by more sensitive methods49. For eight patients with apparently de novo mutations in this cohort, we found no evidence of mosaicism in parents by resequencing with high-depth NGS, but this limited finding does not exclude the possibility of parental mosaicism for families evaluated in the future.\n\nFor those patients who have a tumor with confirmed DICER1 mutation(s), but test negative for germline mutation by Sanger sequencing from blood, it will be important to distinguish as rigorously as possible between tumor-specific, biallelic mutations and the presence of underlying mosaicism. Mutations confined to the tumor will confer no risk for new foci of primary disease in the proband, and family members including potential offspring will be unaffected. Mosaicism, whether for an LOF mutation or an RNase IIIb hotspot mutation, will confer some degree of risk for additional syndromic neoplasias. It may be impossible to unequivocally rule out mosaicism, but techniques such as targeted resequencing by high depth NGS in multiple tissues can greatly improve diagnostic confidence, particularly with respect to RNase IIIb hotspot mutations. For patients who have more than one focus of disease but no germline or mosaic LOF mutation identifiable by targeted NGS of exons, testing for intragenic deletions or larger genomic alterations is recommended.\n\nPatients carrying mosaic RNase IIIb hotspot mutations are predicted, on the basis of both clinical observations and mechanistic rationale, to have extraordinarily high risk as a group for developing multiple disease foci; approaching 100%. It will not be possible to predict individual risk for multifocal disease by allele frequency in blood, as this will not reveal the extent to which other somatic lineages harbor the mutation. Mosaic RNase IIIb hotspot patients will benefit from the most proactive program of family education and surveillance. The International PPB Registry recommends that potential benefits of renal ultrasound and surveillance chest CT be discussed with the family48. The frequency of follow-up chest CTs and chest radiographs should be determined individually, based on patient age, medical history and previous imaging results. Continuing evaluations should include a yearly complete review of systems by a clinician familiar with DICER1 syndrome; yearly screening for ovarian SLCT with review of systems for endocrine dysfunction and pelvic ultrasound for females from early childhood through adulthood; yearly ophthalmologic examination and yearly thyroid examination by palpation and/or ultrasound. Pituitary blastoma and pineoblastoma are rare even in DICER1 syndrome and typically limited to the infant and young child. There is no consensus at this time on screening for intracranial neoplasms.\n\nAs prospective parents, patients who are mosaic for a DICER1 mutation face a theoretical risk for transmitting the mutation of up to 50%, depending upon whether and at what frequency it is present in germ cells. For carriers of a mosaic LOF mutation, the consequences of transmission will be similar to those of a germline LOF mutation carrier. For carriers of a mosaic RNase IIIb mutation, it is uncertain whether transmission could result in a live birth. The absence of RNase IIIb hotspot mutations as inherited alleles in all published studies implies they preclude development to term, but this remains speculative. The mosaic hotspot mutation identified in patient 101 of this cohort was discernable in blood by Sanger sequencing and present at 15% of NGS read counts in normal lymph node tissue (Table 2). Similarly in the two Wilms tumor patients reported by Klein et al. and one pituitary blastoma patient described by De Kock et al., de-novo hotspot mutations were readily detected in blood by Sanger sequencing44,11. Whether the latter case is truly germline or mosaic with high representation in the blood lineage was unclear. Nonetheless, it is clear from these examples that human embryogenesis can tolerate a DICER1 hotspot mutation at high allele frequency in at least some cell lineages. It thus seems possible, though unlikely, that an inherited RNAse IIIb hotspot mutation could be viable.\n\n\nData availability\n\nThe ClinVar accession number(s) for the variant sequences reported in this paper are SCV000195560-SCV000195643.\n\nF1000Research: Dataset 1. Patient information dataset, 10.5256/f1000research.6746.d8076851\n\n\nWeb resources\n\nClinVar database, http://www.ncbi.nlm.nih.gov/clinvar/\n\nOnline Mendelian Inheritance in Man (OMIM), http://www.omim.org/\n\nPPB Genetic Study In: Clinical Trials.Gov available from, http://clinicaltrials.gov/show/NCT00565903\n\nInternational PPB Registry, http://www.ppbregistry.org\n\nNCI DICER1 Phenotype Study, http://dceg.cancer.gov/research/clinical-studies/DICER1-ppb-study", "appendix": "Author contributions\n\n\n\nDAH, YM, GW, LPD, JI and PG conceived the study. DAH, AF, JY, WY, AR designed the experiments. DAH, AF, JY, AR, PS, LD, GW carried out the research. CR, GW, AH, KPS contributed database support and analysis. DRS, MAB and JT provided expertise in genetics. HG performed statistical analysis. DAH and MAB interpreted the results and prepared the first draft of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nInvestigators were supported by NCI R01CA143167 (DAH, LD, CTR, LPD) an American Society of Clinical Oncology Young Investigator Award (LD), The Parson’s Foundation (DAH), Hyundai Hope on Wheels (LD, KAS), a St. Baldrick's fellowship (KAS), The Children’s Discovery Institute at St. Louis Children’s Hospital (DAH) The Hope Street Kids Foundation (DAH), Washington University Department of Pathology and Immunology (DAH, LPD) and St. Louis Children’s Hospital Foundation (DAH). The International PPB Registry is supported by the Pine Tree Apple Tennis Classic, the Theodora H. Lang Charitable Trust, the Children’s Hospitals and Clinics of Minnesota Foundation, and the Randy Shaver Community Cancer Fund. This work was also supported in part by the Hereditary Cancer, Multiplexed Gene Analysis and Tissue Procurement core facilities of the Alvin J. Siteman Cancer Center (NCI Cancer Center Support Grant #P30 CA91842) and by the Division of Cancer Epidemiology and Genetics (DCEG) of the National Cancer Institute Intramural Research Program (DRS).\n\nI confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors thank the families of children with PPB and their many physicians and research associates, who donated time and energy to provide samples for the study.\n\n\nSupplemental data\n\nSupplemental data for ‘Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model’.\n\nSupplemental data file comprises nine tables:\n\nTable S1. DICER1 coding region amplicons for Ion Torrent sequencing\n\nTable S2. DICER1 probes for NanoString copy number assays\n\nTable S3. Summary of germline DICER1 loss-of-function mutations identified in PPB children\n\nTable S4. Germline missense DICER1 mutations (non-hotspot) – additional details\n\nTable S5. Clinical features of children with germline DICER1 mutations\n\nTable S6. Summary of somatic DICER1 RNase IIIb domain “hotspot” mutations identified\n\nTable S7. Sequence results from children with mosaic DICER1 loss of function mutation\n\nTable S8. Sequence results from children with tumor specific, biallelic DICER1 mutations\n\nTable S9. Clinical features of 12 unresolved cases; PPB children who tested negative for germline DICER1 mutation\n\nClick here to access the data.\n\n\nReferences\n\nMessinger YH, Stewart DR, Priest JR, et al.: Pleuropulmonary blastoma: a report on 350 central pathology-confirmed pleuropulmonary blastoma cases by the International Pleuropulmonary Blastoma Registry. Cancer. 2015; 121(2): 276–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPriest JR, McDermott MB, Bhatia S, et al.: Pleuropulmonary blastoma: a clinicopathologic study of 50 cases. Cancer. 1997; 80(1): 147–161. PubMed Abstract \| Publisher Full Text\n\nHill DA, Jarzembowski JA, Priest JR, et al.: Type I pleuropulmonary blastoma: pathology and biology study of 51 cases from the international pleuropulmonary blastoma registry. Am J Surg Pathol. 2008; 32(2): 282–295. PubMed Abstract \| Publisher Full Text\n\nHill DA, Ivanovich J, Priest JR, et al.: DICER1 mutations in familial pleuropulmonary blastoma. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChen Z, Moran K, Richards-Yutz J, et al.: Enhanced sensitivity for detection of low-level germline mosaic RB1 mutations in sporadic retinoblastoma cases using deep semiconductor sequencing. Hum Mutat. 2014; 35(3): 384–391. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKlein S, Lee H, Ghahremani S, et al.: Expanding the phenotype of mutations in DICER1: mosaic missense mutations in the RNase IIIb domain of DICER1 cause GLOW syndrome. J Med Genet. 2014; 51(5): 294–302. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDehner LP, Jarzembowski JA, Hill DA: Embryonal rhabdomyosarcoma of the uterine cervix: a report of 14 cases and a discussion of its unusual clinicopathological associations. Mod Pathol. 2012; 25(4): 602–14. PubMed Abstract \| Publisher Full Text\n\nFoulkes WD, Priest JR, Duchaine TF: DICER1: mutations, microRNAs and mechanisms. Nat Rev Cancer. 2014; 14(10): 662–672. PubMed Abstract \| Publisher Full Text\n\nSamuel N, Villani A, Fernandez CV, et al.: Management of familial cancer: sequencing, surveillance and society. Nat Rev Clin Oncol. 2014; 11(12): 723–31. PubMed Abstract \| Publisher Full Text\n\nSchultz KA, Harris A, Williams GM, et al.: Judicious DICER1 testing and surveillance imaging facilitates early diagnosis and cure of pleuropulmonary blastoma. Pediatr Blood Cancer. 2014; 61(9): 1695–1697. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCampbell IM, Yuan B, Robberecht C, et al.: Parental somatic mosaicism is underrecognized and influences recurrence risk of genomic disorders. Am J Hum Genet. 2014; 95(2): 173–182. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBragg LM, Stone G, Butler MK, et al.: Shining a light on dark sequencing: characterising errors in Ion Torrent PGM Data. PLoS Comput Biol. 2013; 9(4): e1003031. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrenneman M, Field A, Yang J, et al.: Dataset 1 in: Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model. F1000Research. 2015. Data Source" }	[ { "id": "9442", "date": "24 Jul 2015", "name": "Julian A. Martinez-Agosto", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBrenneman et al. present an observational study on a cohort of patients with DICER1 Syndrome. The analysis of 124 probands in combination with available familial data increases the understanding of penetrance and variability of mutations in DICER1 and the effect of these mutations on phenotype. Their identification of additional mosaic, germline and sporadic mutations helps to further elucidate phenotypic differences between these groups and provides insight into disease pathogenesis. Their proposed temporal model of mutation acquisition correlating to disease presentation is supported by the data. However, the following points need clarification: Abstract: On page two the authors state: “A final category of patients lack predisposing germline or mosaic mutations and have disease limited to a single PPB tumor bearing tumor-specific RNase IIIb and LOF mutations.”Cases with a single PPB tumor should not be included under the umbrella of the DICER1 syndrome. A single tumor bearing a causative mutation does not merit inclusion as syndromic, which requires wide spread distribution of mutations in that same gene. This classification is not accurate as these patients simply represent a sporadic neoplasm.Subjects and Methods: Mutation Testing On page three the authors state: “Initial sequencing of blood and saliva DNA samples was by standard Sanger methods described previously or by a commercial laboratory (Ambry Genetics, Aliso Viejo, CA). Low-frequency variants were detected and quantified by targeted next-generation sequenc- ing (NGS) using a custom multiplex PCR panel for DICER1 coding regions (Ion Torrent Ampliseq, Life Technologies, Grand Island, NY, USA). NGS was performed on an Ion Torrent 318 v2 chip (ION PGM Sequencing 200 kit v2, Life Technologies) with an average of 6 samples per chip, to achieve an average depth of coverage of 3000 filtered reads.”Did the authors use the Ion Torrent PCR panel for the analysis of the isolated tumors? This is not mentioned in the methods section. The data presented in Table 2 displays a large distribution in read numbers, which may have implications for data analysis. Can the authors provide an explanation for the wide distribution of read coverage in this table, particularly in the disparity of reads between blood and tumor samples? This should be included in the methods section. Annotation of sequence variants and the spectrum of possible mutations On page three the authors state their methods for annotating variants identified: “For variants assayed by NGS, allele frequencies were calculated from filtered read counts. The SIFT algorithm was used to assess potential significance of novel missense mutations.” It would be helpful, and strengthen the author’s argument that these mutations are pathogenic, to include an analysis of the frequency of loss of function (LOF) and hotspot mutations in the population by determining their minor allele frequency (i.e. using ExAC or 1000 genomes).\n\nNanoString genomic copy number assay On page four the authors state: “Genomic DNA was fragmented and hybridized using the nCounter Prep Station, and hybridization signals quantified using the nCounter Digital Analyzer, according to NanoString’s recommendations.”It is stated in this section that hybridization signals were quantified. However, in table two many of the second hit LOF mutations are simple listed as “allele loss” and not quantified. Can the specific allele region and its quantification be provided as a percentage of allele loss abundance (as was done in table S8 for “Informative SNP” in cases 91,111,112)? Without this data it cannot be determined why the hot spot variant and the allele loss are unequally distributed (cases 104 and 105). Furthermore this would confirm the tumor purity estimates provided as normal cells should not have loss of the second allele. As tumor purity increases so should percentage of allele loss if these mutations are in fact required for tumor formation. As the table reads now it is implied that the second allele loss is complete (50%) in the tumors where it was observed. If this is not the case we ask that the loss be quantified and included, otherwise the “-” should be replaced with “NM” (not measured). Were any positive controls run to confirm the ability to specifically detect copy number events using the Nanostring assay? For example, isolating DNA from preserved tumor samples often yields sheared fragments varying in size, which may hinder probe hybridization across fragments. This may yield false positive allele loss results. Results: Most predisposing DICER1 mutations are inherited loss of function (LOF) mutations On page four the authors state: “Our overall approach to detecting and categorizing predisposing DICER1 mutations in PPB children is shown schematically in Figure 1. We identified germline, heterozygous DICER1 mutations in 90 of the 124 probands in our cohort (72.6%; Table 1, Table S3).”Are the identified LOF variants observed in the general population (ExAC, 1000 Genomes)? On page four the authors state: “In all, 84 of 90 germline DICER1 mutations discovered in patients (93%) truncate the open reading frame before the end of the critical RNase IIIb domain, and are thus predicted to result in complete loss of DICER1 protein function even if the message escapes nonsense-mediated decay.”There is no mention of potential alternative splice isoforms of DICER1, which may be translated despite the presence of early stop and/or frameshift mutations. This is an oversight especially as there is an emerging role for a specific splice variant DICER1e (a splice variant composed of only the RNase IIIa, IIIb and dsRBD domains) in neoplasms. This isoform may utilize a distinct promoter as has been observed for the glucocorticoid receptor gene (Russcher et al., 2007) and not rely on faithful sequence integrity of upstream exons. Two independent reports (Cantini et al., 2014 and Hinkal et al., 2011) have shown increased DICER1e isoforms in oral cancer cells and breast cancer cells respectively. This may be an important factor in discerning potential sub categories of LOF mutations. If in fact DICER1e plays a pathogenic role it is possible that alleles bearing early stop and/or frameshift mutations upstream of the RNaseIIIb domain, which in this study account for 93% of the mutations, are still able to code for this isoform and contribute to disease. The mechanism for oncogenesis may not require a true loss of function first hit but the presence of a modified isoform which in combination with a hot spot mutation would lead to a neomorphic phenotype associated with tumor formation. The authors should acknowledge this possibility in the manuscript. Approximately 10% of predisposing DICER1 mutations are mosaic rather than germline On page six the authors present table 1 “Clinical and Pathologic Features by Predisposing DICER1 Mutation Category.”In table 1 there is a single case reported of a germline LOF mutation and a Wilms tumor (WT). Could the authors speculate on the rarity of WT in their large cohort given the described association of both single hot spot and biallelic mutations in DICER1 with this tumor type (Klein et al., 2014; Wu et al., 2013)? On page eight the authors state: “NGS of tumor tissues from these children identified somatic LOF mutations or allele loss in some but not all specimens, with the caveat that allele loss can be difficult to detect in specimens with low tumor purity (i.e., PPB Type Ir, CN and NCMH).” If the second hit LOF mutation is indeed a “driver” mutation of neoplasm one would expect those mutations to occur early in tumor formation and then clonally expand and be present in a majority of tumor cells. The very difficulty to detect these second hit LOF mutations argues that these tumors are in fact genetically heterogeneous; suggesting that these second hit mutations may represent passenger or modifying mutations but not drivers. The caveat mentioned supports both the authors’ and the alternative hypotheses and this should be included as a possible mechanism of pathogenesis. On page eight the authors state: “Four of the five children with mosaic DICER1 hotspot mutations presented with cystic PPB (type I/IR) rather than sarcomatous disease (type II or type III) and all five have survived to date.”It is surprising that none of the mosaic hot spot cases present with PPB type II/III compared to two thirds of the germline LOF cases. This is not consistent with the more complicated clinical course and numerous neoplasms observed in the mosaic cases. Is it possible that the distribution in the tissue ultimately dictates the severity of the PPB? The authors should discuss as part of their disease model why the mosaic cases have a more complex clinical course while having more benign lung pathology. On page eight the authors state: “Though all five hotspot mosaic children are alive, their clinical experiences have been complicated and arduous (Figure 3).” As the authors state, cases with mosaic DICER1 hot spot mutations present with a complex clinical course. Therefore, including a more detailed clinical description of the five mosaic cases, specifically paying attention to their phenotype, would strengthen this statement. For example, including growth parameters as well as developmental and physical exam findings may help to define this subgroup of the DICER1 syndrome. On page 14 of the supplement a footnote to table S9 mentions “This data set is limited, pursuant to concerns for potential identification of study participants based on particular combinations of clinical and pathologic features. Qualified investigators with specific questions about the study not answered by the data in these tables are invited to contact the International PPB Registry. The Registry will try to accommodate requests for additional data while preserving protected health information.” This concern may be the very reason why more detailed phenotypic data is lacking. However this limits the ability for the reader to draw genotype-phenotype correlations and to compare these cases to those already reported in the literature. We request that at least common clinical findings shared by mosaic cases (if any) are presented in a table without reference to individual cases. This should limit the risk of potential identification of study participants. Table 2On page eight the authors state: “We categorized a DICER1 mutation detected by NGS as mosaic when the following criteria were met: i. The mutation was evidently not a constitutional, germline allele because it was present at sub-heterozygous frequency (arbitrarily taken as below 35% of reads) in peripheral blood and/or other normal tissue samples. ii. The mutation was evidently not specific to a tumor, because the same mutant allele was detected in one or more normal, non-neoplastic tissue samples, OR, the same mutant allele was detected in multiple primary tumors arising in different organs.” It is possible that low abundance mutations detected in blood are not present in a blood cell lineage but may represent metastatic disease (for example in case 102 where the brain tumor is a metastatic event that originated in the lung PPB)? Furthermore cases 102, 103, 104, and 105 have very low mutation abundance in blood with cases 103-105 carrying less than 0.3%. These low numbers are perhaps evidence of unrecognized metastatic disease in these patients, the detection of which has been previously described (Haber and Velculescu, 2014). Testing of more “normal” tissues is needed for fulfilling the mosaicism criteria as proposed by the authors. It is more likely that the cases in table 2 have lower levels of mosaicism limited to a small number of tissues in contrast to other cases with more widely distributed hot spot mutations (Klein et al., 2014). Additional phenotypic clinical data for these cases (as requested above) is needed to properly make a comparison. Tumor purity is very low in mosaic cases samples (Table 2), and this is used as an argument for why second hit mutations are lower in abundance. However, it is also possible that the second hit mutation is unequally distributed throughout the tumor and in fact absent from some regions of the tumor. This possibility should be mentioned in the manuscript. Tumor-specific, biallelic DICER1 mutations account for about 10% of PPB cases On page eight the authors state: “In twelve children, we identified biallelic DICER1 mutations present at high allele frequencies in a PPB tumor, but not detectable in blood even with the benefit of high-depth NGS (Table S8).”These cases likely represent sporadic neoplasm mutations. We question if these cases should be classified as having the “DICER1 syndrome.” A more clear distinction between isolated PPB and the “DICER1 Syndrome” should be included. Currently unresolved cases On page eight the authors state: “Twelve PPB probands in our cohort are negative for predisposing DICER1 mutations detectable in blood DNA by Sanger sequencing or NGS of coding exons.”The authors include an additional 12 unresolved cases (Table S9). We suggest that a potential etiology for cases in which DICER1 mutations are absent would be caused by mutations in DROSHA or other genes involved in the microRNA processing pathway. It has been established that at least in the pathogenesis of sporadic Wilms tumor RNaseIIIb mutations in DROSHA phenocopy those in DICER1, although possibly by distinct mechanisms (Rakheja, 2014). While the authors pursued DROSHA testing via Sanger sequencing exclusively in blood (Table S9 Footnote: “Sanger sequencing in blood DNA for DROSHA, XPO5, and the DICER1 promoter region”) this approach might have missed low abundance mosaic mutations. Furthermore, some of these “unresolved” cases (121, 123 and 124) have been identified as carrying hot spot mutations in their tumors without the presence of a second hit. This may illustrate that the hotspot mutations alone may be sufficient for tumorogenesis. On page 5 the authors present Figure 1 “Study Design” After reviewing this cohort we call into question the designation of “unresolved” in one case. We would like to propose that case 123 is actually mosaic due to the presence of the same hotspot mutation in two distinct disease foci. This case cannot be excluded as mosaic based on the absence of the mutation in blood as it is similar to case 105 where the sequencing from blood is not above the error threshold. Case 123 should therefore be moved to table two, and the study design/mosaic criteria amended to classify case 105 and 123 as mosaic even in the absence of the hotspot mutation in normal tissue. The absence of a LOF second hit in the presence of a mosaic hot spot mutation should represent a distinct subset of mosaic cases and not classified as “unresolved.” Discussion: Genotype-phenotype correlation of predisposing mutations in PPB-DICER1 syndrome On page ten the authors state: “All germline DICER1 truncating mutations are predicted to be essentially equivalent in their effect: complete loss of function in miRNA processing.”As mentioned above this conclusion must be tempered by the possibility of an alternatively spliced variant of DICER1 that could be expressed despite a truncating mutation upstream of the RNase IIIb domain.\n\nOn page ten the authors state: “Neomorphic RNase IIIb domain function (skewed 5p/3p miRNA production) is a recurring feature of DICER1 tumors, and it is plausible that loss of all wildtype RNase IIIb function is required for it to become tumorigenic.”The statement “loss of all wildtype RNase IIIb function is required for it to become tumorigenic” does not apply to all categories of the DICER1 syndrome. From the data as it is presented in this study the only category where this can be concluded is from the sporadic tumors, which are distinct from the DICER1 syndrome. In these tumors it is clear from the data in table S8 that all hot spot mutations are accompanied by LOF mutations with corresponding abundances, which are certainly a characteristic of these aggressive lung neoplasms. However, we cannot conclude causality for the second hit mutations in all DICER1 tumors since (1) in the mosaic hot spot cases (Table 2) the observed abundance for the second LOF hit mutations is always less than the hot spot mutation and (2) there are cases of tumors in this report that lack a second hit (Case 105: NCMH, Case 123 CN and PPB). A main objection to the analysis and interpretation of these results is the lack of an explanation for the differences in mutation abundance between LOF and hot spot mutations within a tumor and the presence of tumors without a second hit. This raises questions as to whether the LOF mutations are in fact drivers of tumorigenesis or passenger mutations. Although tumor purity could be partially responsible for these inconsistencies, the data on its own does not sufficiently establish that these LOF mutations are required for tumor formation in non-sporadic tumor cases when they are not present in all neoplastic cells. In the model as it is proposed by the authors, cases that are mosaic for RNaseIIIb mutations display no clinical findings until a second LOF mutation occurs which drives and is essential for tumor formation (Page 11, last paragraph). We believe there is the possibility for another explanation. A single RNase IIIb mutation alone could have a pro-onocogenic effect on distinct cell types at specific developmental stages. As tumorigenesis proceeds, a LOF mutation in the other allele may arise as the tumor drifts, further aggravating the 5p/3p imbalance in a sub population of tumor cells. Supporting this alternative model, cases 105, 121, 123 and 124 are reported to have neoplasms with no second hit detected. If this second hit is essential why do these tumors lack the LOF second hits? In aggregate there are 4 mosaic cases (2 in this report and 2 in the literature), which, in combination with the absence of germline true heterozygote hot spot mutations, support the alternate model that mosaic hot spot mutations are likely pathogenic on their own and the authors should expand their model to include this alternate explanation. Supplemental DataTable S5 Clinical features of children with germline DICER1 mutations Can the authors comment on why mortality is higher in germline LOF mutation carriers than it is in the mosaic “hot spot” mutation carriers even though the latter have a more complicated clinical course? Could this be due to the association of PPB type I/IR with mosaic cases and PPB type II and III with germline LOF cases? Minor ChangesThe authors should include appropriate references to any manuscripts in which any of these PPB registry cases have been previously reported. While not essential, it would be informative to include any affected siblings for the 10 identified de novo LOF cases to support or refute potential germline mosaicism in the parents (Page 4)?Table 1 includes a single case of a germline LOF mutation and a Wilms tumor (WT). However, little else is described about this case. Furthermore there is no mention of this case in the supplementary materials. Please include mutation analysis and additional phenotypic information for this case.", "responses": [ { "c_id": "3329", "date": "12 Jan 2018", "name": "D. Ashley Hill", "role": "Author Response", "response": "Author Responses in Bold to Referee Report Julian A. Martinez-Agosto, Departments of Pediatrics and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, CA, USA Steven Klein, Departments of Pediatrics and Human Genetics, David Geffen School of Medicine, University of California, Los Angeles, USA Approved with Reservations Brenneman et al. present an observational study on a cohort of patients with DICER1 Syndrome. The analysis of 124 probands in combination with available familial data increases the understanding of penetrance and variability of mutations in DICER1 and the effect of these mutations on phenotype. Their identification of additional mosaic, germline and sporadic mutations helps to further elucidate phenotypic differences between these groups and provides insight into disease pathogenesis. Their proposed temporal model of mutation acquisition correlating to disease presentation is supported by the data. However, the following points need clarification: Abstract: On page two the authors state: “A final category of patients lack predisposing germline or mosaic mutations and have disease limited to a single PPB tumor bearing tumor-specific RNase IIIb and LOF mutations.” Cases with a single PPB tumor should not be included under the umbrella of the DICER1 syndrome. A single tumor bearing a causative mutation does not merit inclusion as syndromic, which requires wide spread distribution of mutations in that same gene. This classification is not accurate as these patients simply represent a sporadic neoplasm. Right. We don’t consider these to be cases of DICER1 syndrome, and didn’t actually refer to them as such in the article. But we didn’t think to explicitly state the distinction between sporadic and syndromic PPB. We should have, and that has been corrected, both in the abstract and the Results section. This was a prospective study of PPB, so in our report we have accounted for all cases of PPB that were accrued for sequencing, whether they turned out to meet criteria for DICER1 syndrome or not. The title of the article has also been revised to reflect our use of PPB as the basis for accrual. It may still imply to some readers that all cases being described are DICER1 syndrome, but we can’t think of a way to avoid that without making the title absurdly long. Subjects and Methods: Mutation Testing On page three the authors state: “Initial sequencing of blood and saliva DNA samples was by standard Sanger methods described previously or by a commercial laboratory (Ambry Genetics, Aliso Viejo, CA). Low-frequency variants were detected and quantified by targeted next-generation sequenc- ing (NGS) using a custom multiplex PCR panel for DICER1 coding regions (Ion Torrent Ampliseq, Life Technologies, Grand Island, NY, USA). NGS was performed on an Ion Torrent 318 v2 chip (ION PGM Sequencing 200 kit v2, Life Technologies) with an average of 6 samples per chip, to achieve an average depth of coverage of 3000 filtered reads.” Did the authors use the Ion Torrent PCR panel for the analysis of the isolated tumors? This is not mentioned in the methods section. The data presented in Table 2 displays a large distribution in read numbers, which may have implications for data analysis. Can the authors provide an explanation for the wide distribution of read coverage in this table, particularly in the disparity of reads between blood and tumor samples? This should be included in the methods section. All sequencing of tumors was done on the Ion Torrent platform, using the panel of PCR amplicons for DICER1 coding regions detailed in Supplemental Data (Table S1). The variation in total read numbers apparent in Table 2 is related to several factors: the number of sequencing runs done for a sample, the number of reads per run that pass quality metrics, the “purity” of tumor specimens and the occurence of allele loss in some. For some blood samples, fewer samples were loaded per chip, or multiple sequencing runs were done, to get higher total read numbers and improve the sensitivity of mutation detection. This was so particularly in cases where initial Sanger sequencing of blood was negative, but we had reason to suspect a mosaic mutation might be present at low allele frequency (i.e., a mutation had already been detected in more than one tissue/tumor specimen). Annotation of sequence variants and the spectrum of possible mutations On page three the authors state their methods for annotating variants identified: “For variants assayed by NGS, allele frequencies were calculated from filtered read counts. The SIFT algorithm was used to assess potential significance of novel missense mutations.” It would be helpful, and strengthen the author’s argument that these mutations are pathogenic, to include an analysis of the frequency of loss of function (LOF) and hotspot mutations in the population by determining their minor allele frequency (i.e., using ExAC or 1000 genomes). None of the specific LOF or hotspot variants we identified in this study have been reported as germline alleles in the 1000 Genomes data base, but that’s beside the point really. We know that many of the LOF alleles in our cohort occur in people who have never been diagnosed with any form of syndromic disease, because there are many confirmed but asymptomatic carriers identified among family members in the PPB Registry. In other words, the penetrance of DICER1 mutations in pedigrees is low. So, unfortunately, population data isn’t very helpful in establishing the pathogenicity of individual mutations. NanoString genomic copy number assay On page four the authors state: “Genomic DNA was fragmented and hybridized using the nCounter Prep Station, and hybridization signals quantified using the nCounter Digital Analyzer, according to NanoString’s recommendations.” It is stated in this section that hybridization signals were quantified. However, in table two many of the second hit LOF mutations are simply listed as “allele loss” and not quantified. Can the specific allele region and its quantification be provided as a percentage of allele loss abundance (as was done in table S8 for “Informative SNP” in cases 91,111,112)? Without this data it cannot be determined why the hot spot variant and the allele loss are unequally distributed (cases 104 and 105). Furthermore this would confirm the tumor purity estimates provided as normal cells should not have loss of the second allele. As tumor purity increases so should percentage of allele loss if these mutations are in fact required for tumor formation. As the table reads now it is implied that the second allele loss is complete (50%) in the tumors where it was observed. If this is not the case we ask that the loss be quantified and included, otherwise the “-” should be replaced with “NM” (not measured). Were any positive controls run to confirm the ability to specifically detect copy number events using the Nanostring assay? For example, isolating DNA from preserved tumor samples often yields sheared fragments varying in size, which may hinder probe hybridization across fragments. This may yield false positive allele loss results. In this project, we used the NanoString Copy Number Assay only to interrogate genomic DNA extracted from blood for DICER1 exonic deletions in a handful of cases. The Subjects and Methods section has been amended to make this clear. For detection of exon deletions in blood DNA, our NanoString controls consisted of blood DNA from children with normal diploid DICER1 copy number, and DNA from one PPB tumor with three copies of DICER1 as determined by next generation sequencing (Pugh et al, 2014). We did not use NanoString to evaluate DICER1 copy number in tumors. In our limited experience, we found that even slight degradation of DNA in FFPE tumor specimens makes reliable detection of copy number changes with NanoString very challenging. Admixture of tumor and normal cells makes it more so. Results Most predisposing DICER1 mutations are inherited loss of function (LOF) mutations On page four the authors state: “Our overall approach to detecting and categorizing predisposing DICER1 mutations in PPB children is shown schematically in Figure 1. We identified germline, heterozygous DICER1 mutations in 90 of the 124 probands in our cohort (72.6%; Table 1, Table S3).” Are the identified LOF variants observed in the general population (ExAC, 1000 Genomes)? Please see our response above, in relation to Annotation of sequence varaints. On page four the authors state: “In all, 84 of 90 germline DICER1 mutations discovered in patients (93%) truncate the open reading frame before the end of the critical RNase IIIb domain, and are thus predicted to result in complete loss of DICER1 protein function even if the message escapes nonsense-mediated decay.” There is no mention of potential alternative splice isoforms of DICER1, which may be translated despite the presence of early stop and/or frameshift mutations. This is an oversight especially as there is an emerging role for a specific splice variant DICER1e (a splice variant composed of only the RNase IIIa, IIIb and dsRBD domains) in neoplasms. This isoform may utilize a distinct promoter as has been observed for the glucocorticoid receptor gene (Russcher et al., 2007) and not rely on faithful sequence integrity of upstream exons. Two independent reports (Cantini et al., 2014 and Hinkal et al., 2011) have shown increased DICER1e isoforms in oral cancer cells and breast cancer cells respectively. This may be an important factor in discerning potential subcategories of LOF mutations. If in fact DICER1e plays a pathogenic role it is possible that alleles bearing early stop and/or frameshift mutations upstream of the RNaseIIIb domain, which in this study account for 93% of the mutations, are still able to code for this isoform and contribute to disease. The mechanism for oncogenesis may not require a true loss of function first hit but the presence of a modified isoform which in combination with a hot spot mutation would lead to a neomorphic phenotype associated with tumor formation. The authors should acknowledge this possibility in the manuscript. DICER1e is intriguing but still a very early story. It’s not known whether or how this isoform participates in miRNA processing or other possible DICER1 functions. No specific mechanism by which differential expression of a DICER1e protein isoform might contribute to tumorigenesis has been established. It’s also not yet known whether the DICER1e mRNA is an alternate splice product or an independent transcript from a different promoter. It’s not clear whether or how truncating mutations in upstream exons influence expression of the DICER1e mRNA. And unfortunately, our data includes no information about whether a DICER1e mRNA or protein was expressed in any of the tumors of patients who were included in this study. As we have nothing to support it, we have chosen not to speculate about DICER1e in this report. But hypothetically, let’s assume that: i.) DICER1e has distinct activities that can contribute to tumorigenesis, ii.) DICER1e can be expressed regardless of mutations in upstream exons, and iii.) expression of DICER1e, rather than loss of full-length DICER1, is the important co-determinant of tumorigenesis when combined with an RNase IIIb hotspot mutation. We then have to explain why germline mutations in exons 21-27 (n = 46, Figure 3), which presumably would inactivate DICER1e, seem to correlate with disease just as strongly as mutations in exons 2-18 (n = 44), which would not. Suggestions? Approximately 10% of predisposing DICER1 mutations are mosaic rather than germline On page six the authors present Table 1. Clinical and Pathologic Features by Predisposing DICER1 Mutation Category. In Table 1, there is a single case reported of a germline LOF mutation and a Wilms tumor (WT). Could the authors speculate on the rarity of WT in their large cohort given the described association of both single hot spot and biallelic mutations in DICER1 with this tumor type (Klein et al., 2014; Wu et al., 2013)? In addition to the two remarkable cases of Wilms tumor with DICER1 mutation described in your own paper, and those in Wu et al, 2013, we’re aware of the cases published by Rakeja et al, 2014 and Torrezan et al, 2014. The low incidence of Wilms tumor in the present cohort may reflect some ascertainment bias. The criterion for inclusion in this cohort was a diagnosis of pleuropulmonary blastoma (PPB). But there have been children with other DICER1-associated tumors in whom PPB was not diagnosed first (or at all). Ascertainment bias might also pertain to other DICER1-associated tumors that are rare or absent in our cohort, i.e., pituitary blastoma. That said, the frequency of Wilms tumor in this cohort roughly parallels what we have seen among PPB children and their extended families in the International PPB Registry more generally, i.e., it is low (about 1%), similar to pineoblastoma and pituitary blastoma. In our experience, cystic nephroma is far more common than Wilms tumor or Wilms precursor lesions (nephrogenic rests, nephroblastomatosis) in DICER1 syndrome. Cystic nephroma can progress to primitive renal sarcoma, which has some morphologic overlap with Wilms tumor, but clearly there are also classic-morphology Wilms tumors with DICER1 mutations. How the genetic etiologies of these neoplasias converge or diverge is yet to be discovered. On page eight the authors state: “NGS of tumor tissues from these children identified somatic LOF mutations or allele loss in some but not all specimens, with the caveat that allele loss can be difficult to detect in specimens with low tumor purity (i.e., PPB Type Ir, CN and NCMH).” If the second hit LOF mutation is indeed a “driver” mutation of neoplasm one would expect those mutations to occur early in tumor formation and then clonally expand and be present in a majority of tumor cells. The very difficulty to detect these second hit LOF mutations argues that these tumors are in fact genetically heterogeneous; suggesting that these second hit mutations may represent passenger or modifying mutations but not drivers. The caveat mentioned supports both the authors’ and the alternative hypotheses and this should be included as a possible mechanism of pathogenesis. The passage you cite has been revised to note that specific LOF mutations or evidence of allele loss were found in all tumor specimens sequenced for hotspot mosaic patients. Please see the revised Table 2. Please also see our response to Drs. Chen and Amatruda, which includes an extended discussion of how allele loss has been inferred from NGS read counts, with examples. We agree with you that at least some of the tumors described in Table 2 must be genetically heterogeneous. The tumor specimens sequenced for patient #103 are the clearest examples. We also agree that the DICER1 LOF mutations we detected should not be considered driver mutations. The term driver mutation is usually reserved for gain-of-function events causing activation or overexpression of oncogenes, such as KRAS or MYC, that stimulate (“drive”) tumor cell growth and division. It’s not just a matter of labels. Continued expression of an activated oncogene is subject to positive selection during expansion of a tumor cell population. But for a tumor suppressor gene, a specific loss-of-function change in coding sequence will not always be retained by selective pressure. Even if continuing absence of expression is needed for growth after initiation of a tumor, any kind of subsequent LOF mutational event can suffice to maintain it: complete or partial deletion of the gene, translocations or inversions that disrupt the open reading frame, copy-neutral loss of heterozygosity through gene conversion or mitotic crossover, complete or partial chromosome loss, etc. Many of these events will not be detected by exon sequencing. Thus, some level of heterogeneity with regard to LOF mutations should not come as a surprise; neither should low allele frequency of a specific LOF coding sequence change, nor the absence of such changes in some tumor cell populations. The question of why specific LOF mutations are detected at low allele frequencies (or not at all) in some tumors of hotspot mosaic children, and what the true significance of LOF mutations in tumorigenesis may be, has also attracted the attention of our other two reviewers, Kenneth Chen and Jim Amatruda. They also suggest an alternative hypothesis, which may be similar to what you have in mind. Please take a look at our response to Drs. Chen and Amatruda and at the revised Discussion section, in which we now explicitly raise the possibility of a dominant-negative effect for hotspot mutations, in some tissues at least. We look forward to discussing this further with you in the future. On page eight the authors state: “Four of the five children with mosaic DICER1 hotspot mutations presented with cystic PPB (type I/IR) rather than sarcomatous disease (type II or type III) and all five have survived to date.” It is surprising that none of the mosaic hotspot cases present with PPB type II/III compared to two thirds of the germline LOF cases. This is not consistent with the more complicated clinical course and numerous neoplasms observed in the mosaic cases. Is it possible that the distribution in the tissue ultimately dictates the severity of the PPB? The authors should discuss as part of their disease model why the mosaic cases have a more complex clinical course while having more benign lung pathology. One of the hotspot mosaic children had both PPB type II and type Ir; this was noted in Table 1. That patient (study ID# 102) recently died of metastatic PPB. Another patient now recategorized as hotspot mosaic (study ID# 120) also had both PPB type II and type Ir. Even so, incidence of advanced PPB has been low among the hotspot mosaic children as a group, and we also found this surprising. But it’s not clear whether hotspot mosaic children actually have less aggressive lung pathology, or simply tend to get treated earlier, before it progresses. All the children in this group developed bilateral, multifocal lung cysts, and at very early ages, similarly to the two mosaic cases you described. Their early diagnosis was no doubt in part because their lung lesions, though type I or Ir, were numerous and/or large enough to cause obvious breathing difficulty. For children who have germline LOF mutations this is not always the case. They often develop only a single PPB lesion in one lung, and as a group tend to be diagnosed later (Table 1). This means the lesions have more time to progress from type I to type II or III, contributing to higher incidence of advanced PPB and metastatic disease at diagnosis and greater mortality. The possibility you suggest, that distribution of the mutation might influence not just the number of lesions but also their severity, is not one we thought of before. Nothing in our data rules that out. On page eight the authors state: “Though all five hotspot mosaic children are alive, their clinical experiences have been complicated and arduous (Figure 3).” As the authors state, cases with mosaic DICER1 hot spot mutations present with a complex clinical course. Therefore, including a more detailed clinical description of the five mosaic cases, specifically paying attention to their phenotype, would strengthen this statement. For example, including growth parameters as well as developmental and physical exam findings may help to define this subgroup of the DICER1 syndrome. A footnote to table S9 mentions “This data set is limited, pursuant to concerns for potential identification of study participants based on particular combinations of clinical and pathologic features. Qualified investigators with specific questions about the study not answered by the data in these tables are invited to contact the International PPB Registry. The Registry will try to accommodate requests for additional data while preserving protected health information.” This concern may be the very reason why more detailed phenotypic data is lacking. However this limits the ability for the reader to draw genotype-phenotype correlations and to compare these cases to those already reported in the literature. We request that at least common clinical findings shared by mosaic cases (if any) are presented in a table without reference to individual cases. This should limit the risk of potential identification of study participants. All the shared characteristics of the hotspot mosaic children that we know of have been presented in the figures and tables, and further described in the text. Figure 3 presents all syndromic disease and age at each diagnosis individually for each patient. Table 1 provides a summary of all syndromic disease for the hotspot mosaic children as a group, and statistical comparisons to other categories. We understand your particular interest in body growth metrics and developmental milestones, and regret we cannot furnish them. But we don’t have detailed physical exam information for the seven children ultimately identified as hotspot mosaic. This was a prospective study conducted at multiple centers. Participating clinicians didn’t report that information for all probands over the course of this study, probably because they weren’t asked to. In the past, abnormalities of growth and developmental delays had generally not been perceived as features of familial PPB/DICER1 syndrome, and the mosaic hotspot phenomenon was only identified in retrospect. Table 2 On page eight the authors state: “We categorized a DICER1 mutation detected by NGS as mosaic when the following criteria were met: i. The mutation was evidently not a constitutional, germline allele because it was present at sub-heterozygous frequency (arbitrarily taken as below 35% of reads) in peripheral blood and/or other normal tissue samples. ii. The mutation was evidently not specific to a tumor, because the same mutant allele was detected in one or more normal, non-neoplastic tissue samples, OR, the same mutant allele was detected in multiple primary tumors arising in different organs.” It is possible that low abundance mutations detected in blood are not present in a blood cell lineage but may represent metastatic disease (for example in case 102 where the brain tumor is a metastatic event that originated in the lung PPB)? Furthermore cases 102, 103, 104, and 105 have very low mutation abundance in blood with cases 103-105 carrying less than 0.3%. These low numbers are perhaps evidence of unrecognized metastatic disease in these patients, the detection of which has been previously described (Haber and Velculescu, 2014). Testing of more “normal” tissues is needed for fulfilling the mosaicism criteria as proposed by the authors. It is more likely that the cases in table 2 have lower levels of mosaicism limited to a small number of tissues in contrast to other cases with more widely distributed hot spot mutations (Klein et al., 2014). Additional phenotypic clinical data for these cases (as requested above) is needed to properly make a comparison. It’s conceivable the very small numbers of hotspot mutation reads in blood samples from patients 103, 104 and 105 were from tumor cells shed into the circulation (the 4.6% frequency in patient 102 seems too high for that). Regardless of their origin, we didn’t consider those blood reads reliable evidence of mosaicism, and based no conclusions upon them. Our conclusion in favor of mosaicism in patients 103, 104 and 105 (and the patient recategorized in revision, ID# 123) is based upon presence of the same hotspot mutation in multiple primary tumors and/or non-neoplastic tissues. Certainly it would help to have more normal tissue specimens to sequence, but none were available and we can hardly request medically unnecessary biopsies to improve our data set. We stand by our conclusion of mosaicism, because it seems to us far more plausible than the only other explanation possible: that the same somatic hotspot mutation occurred independently in two to five different tissue and/or tumor sites. Tumor purity is very low in mosaic cases samples (Table 2), and this is used as an argument for why second hit mutations are lower in abundance. However, it is also possible that the second hit mutation is unequally distributed throughout the tumor and in fact absent from some regions of the tumor. This possibility should be mentioned in the manuscript. Yes, tumor purity was generally lower in specimens from hotspot mosaic children than other patient categories, probably due at least in part to the greater proportion of non-PPB tumors encountered. Some syndromic lesions such as PPB type I/Ir, cystic nephroma and juvenile-type polyps of the small intestine have complicated histo-pathological features, comprising mixtures of neoplastic and non-neoplastic cell types. We agree that at least some of the LOF mutations reported in Table 2 must be unevenly distributed in the tumors specimens sequenced, particularly for study ID# 103. We didn’t cite low tumor purity as a reason for differences between hotspot and LOF allele frequencies, but it does make determinations of allele loss less certain, and the two things are related, as discussed in our responses above. Tumor-specific, biallelic DICER1 mutations account for about 10% of PPB cases On page eight the authors state: “In twelve children, we identified biallelic DICER1 mutations present at high allele frequencies in a PPB tumor, but not detectable in blood even with the benefit of high-depth NGS (Table S8).” These cases likely represent sporadic neoplasm mutations. We question if these cases should be classified as having the “DICER1 syndrome.” A more clear distinction between isolated PPB and the “DICER1 Syndrome” should be included. As noted above in relation to the abstract, we concur that sporadic PPB should be distinguished from DICER1 syndrome. The paragraph you cite has been revised to make this clear. Currently unresolved cases On page eight the authors state: “Twelve PPB probands in our cohort are negative for predisposing DICER1 mutations detectable in blood DNA by Sanger sequencing or NGS of coding exons.” The authors include an additional 12 unresolved cases (Table S9). We suggest that a potential etiology for cases in which DICER1 mutations are absent would be caused by mutations in DROSHA or other genes involved in the microRNA processing pathway. It has been established that at least in the pathogenesis of sporadic Wilms tumor, RNaseIIIb mutations in DROSHA phenocopy those in DICER1, although possibly by distinct mechanisms (Rakheja, 2014). While the authors pursued DROSHA testing via Sanger sequencing exclusively in blood (Table S9 Footnote: “Sanger sequencing in blood DNA for DROSHA, XPO5, and the DICER1 promoter region”) this approach might have missed low abundance mosaic mutations. Furthermore, some of these “unresolved” cases (121, 123 and 124) have been identified as carrying hot spot mutations in their tumors without the presence of a second hit. This may illustrate that the hotspot mutations alone may be sufficient for tumorogenesis. After reviewing this cohort we call into question the designation of “unresolved” in one case. We would like to propose that case 123 is actually mosaic due to the presence of the same hotspot mutation in two distinct disease foci. This case cannot be excluded as mosaic based on the absence of the mutation in blood as it is similar to case 105 where the sequencing from blood is not above the error threshold. Case 123 should therefore be moved to table two, and the study design/mosaic criteria amended to classify case 105 and 123 as mosaic even in the absence of the hotspot mutation in normal tissue. The absence of a LOF second hit in the presence of a mosaic hot spot mutation should represent a distinct subset of mosaic cases and not classified as “unresolved.” The 12 cases that were detailed in supplemental Table S9 are the 12 cases mentioned in the sentence you quote, not additional. The sentence has been revised to make that clear. Table S9 has become Table S10 in the revised paper, and now presents only 10 unresolved cases. With regard to other genetic etiologies, yes, the limited genomic sequencing we did might easily have missed many kinds of mutations in DROSHA or XPO5. And there are additional candidate genes we didn’t look at. To us it seems quite possible that mutations in other genes of the miRNA processing pathway might phenocopy DICER1 mutation in PPB or additional neoplasms besides Wilms tumor. Perhaps it’s just a matter of time before solid evidence of that turns up. Your assessment of patient 123 is correct. Since the original submission of this report, additional sequencing results have given us the confidence to call hotspot mosaicism, and patient 123 now appears in Table 2 and Figure 3. We found no LOF mutation by Ion Torrent sequencing of the coding exons, but the hotspot allele frequencies obtained from read counts suggest loss of the second DICER1 allele in the two tumor specimens sequenced. Please see the extended discussion of how allele loss was inferred in our response to Drs. Chen and Amatruda. After we posted this report, the Foulkes lab published an overlapping one describing three of the same DICER1 patients: study ID#s 102, 105 and 120 (Patients 1, 2 and 4, respectively, in their report). They propose that patient 120 is also hotspot mosaic, but with very limited tissue distribution of the hotspot mutation, possibly confined to the lungs. We concur. But take a look and draw your own conclusions: de Kock et al, Journal of Medical Genetics, doi: 10.1136/jmedgenet-2015-103428. Patient 120 also now appears in Table 2 and Figure 3. Discussion Genotype-phenotype correlation of predisposing mutations in PPB-DICER1 syndrome On page ten the authors state: “All germline DICER1 truncating mutations are predicted to be essentially equivalent in their effect: complete loss of function in miRNA processing.” As mentioned above this conclusion must be tempered by the possibility of an alternatively spliced variant of DICER1 that could be expressed despite a truncating mutation upstream of the RNase IIIb domain. On page ten the authors state: “Neomorphic RNase IIIb domain function (skewed 5p/3p miRNA production) is a recurring feature of DICER1 tumors, and it is plausible that loss of all wildtype RNase IIIb function is required for it to become tumorigenic.” The statement “loss of all wildtype RNase IIIb function is required for it to become tumorigenic” does not apply to all categories of the DICER1 syndrome. From the data as it is presented in this study the only category where this can be concluded is from the sporadic tumors, which are distinct from the DICER1 syndrome. In these tumors it is clear from the data in table S8 that all hot spot mutations are accompanied by LOF mutations with corresponding abundances, which are certainly a characteristic of these aggressive lung neoplasms. However, we cannot conclude causality for the second hit mutations in all DICER1 tumors since (1) in the mosaic hot spot cases (Table 2) the observed abundance for the second LOF hit mutations is always less than the hot spot mutation and (2) there are cases of tumors in this report that lack a second hit (Case 105: NCMH, Case 123 CN and PPB). A main objection to the analysis and interpretation of these results is the lack of an explanation for the differences in mutation abundance between LOF and hot spot mutations within a tumor and the presence of tumors without a second hit. This raises questions as to whether the LOF mutations are in fact drivers of tumorigenesis or passenger mutations. Although tumor purity could be partially responsible for these inconsistencies, the data on its own does not sufficiently establish that these LOF mutations are required for tumor formation in non-sporadic tumor cases when they are not present in all neoplastic cells. In the model as it is proposed by the authors, cases that are mosaic for RNaseIIIb mutations display no clinical findings until a second LOF mutation occurs which drives and is essential for tumor formation (Page 11, last paragraph). We believe there is the possibility for another explanation. A single RNase IIIb mutation alone could have a pro-onocogenic effect on distinct cell types at specific developmental stages. As tumorigenesis proceeds, a LOF mutation in the other allele may arise as the tumor drifts, further aggravating the 5p/3p imbalance in a sub population of tumor cells. Supporting this alternative model, cases 105, 121, 123 and 124 are reported to have neoplasms with no second hit detected. If this second hit is essential why do these tumors lack the LOF second hits? In aggregate there are 4 mosaic cases (2 in this report and 2 in the literature), which, in combination with the absence of germline true heterozygote hot spot mutations, support the alternate model that mosaic hot spot mutations are likely pathogenic on their own and the authors should expand their model to include this alternate explanation. As mentioned above, we have revised the Discussion to note the model we present has apparent exceptions, and that loss of wild-type DICER1 function may not to be required in order for a hotspot mutation to instigate tumor formation in all tissues or circumstances. We point out the need to consider an alternative model, in which RNase IIIb-mutant DICER1 exerts a dominant-negative effect over the wild-type protein. We thank all of our reviewers for encouraging us to broach this possibility. At the time we first submitted the paper, we had decided against any mention of a dominant-negative model in our Discussion, because it seemed too speculative and we had no data of our own to directly support it. We also favor the possibility, suggested by Drs. Amatruda and Chen, that DICER1 hotspot mutations may introduce a genome stability defect, by compromising DNA repair through as-yet-unidentified mechaisms, but chose not to mention that in the paper either, for the same reasons. But despite our general agreement, we must take exception to some of the ideas you express above. Regarding the model we described, in which both a neomorphic DICER1 hotspot mutation and loss of wild-type DICER1 function are usually required to promote tumorigenesis, you declare that “…the only category where this can be concluded is from the sporadic tumors, which are distinct from the DICER1 syndrome.” This is simply not the case. Please consider again the features of germline mutation patients. All have LOF mutations, either inherited or de novo, present at heterozygous frequency in blood or other normal tissues sampled and presumed to be present in all cells of their bodies. In all PPBs and in nearly every other tumor of these patients from which DNA of reasonable quality has been recovered and sequenced, a hotspot missense mutation was also discovered (the most conspicuous exception being the series of pineoblastomas described by the Foulkes lab, in which DICER1 function appears to have been lost entirely). If it were true that DICER1-associated tumorigenesis in the lung and most other vulnerable tissues does not usually require loss of wild-type DICER1 function, there would be no familial PPB/DICER1 syndrome. Similarly, children with mosaic LOF mutations would not have emerged as a category of PPB patients if LOF mutations did not impart susceptibility. The sporadic cases, in which both a hotspot mutation and LOF mutation or allele loss are confined to a single tumor, can be viewed as the exceptions that prove the rule. If concurrent loss of wild type DICER1 function is not typically necessary for a hotspot missense mutation to instigate tumorigenesis, why do they occur together even in non-syndromic PPB? Regarding sequencing results in children with mosaic hotspot mutations: You interpret low allele frequencies of LOF mutations found in some tumor specimens, and failure to detect LOF mutations in some tumor specimens, as positive evidence that loss of wild type DICER1 function was not neccesary for tumorigenesis. But it is not positive evidence for anything beyond the inherent limitations of exon sequencing. You implicitly recognize a role for genomic instability when you suggest that LOF mutations present at low allele frequency may be only passenger mutations. Surely you can accept that ongoing genomic instability in tumor cell populations might also manifest as allele loss? The heart of the problem is this: failure to discover a specific LOF mutation in a tumor by DICER1 exon sequencing does not establish that wild type DICER1 protein is still being expressed. If there is ever to be confirmation of our shared hypothesis that a DICER1 hotspot mutation can be sufficient to cause tumorigenesis in some settings, it will take something more than arguments based on LOF allele frequencies. Table S5. Clinical features of children with germline DICER1 mutations Can the authors comment on why mortality is higher in germline LOF mutation carriers than it is in the mosaic “hotspot” mutation carriers even though the latter have a more complicated clinical course? Could this be due to the association of PPB type I/IR with mosaic cases and PPB type II and III with germline LOF cases? Please see our response, above, to your queries about the section of Results describing hotspot mosaic children. As mentioned, two of the (now seven) patients identified as hotspot mosaic had PPB type II, and one has since died of metastatic disease. But for other reasons as well, it’s not clear to us whether mortality actually is lower in the mosaic hotspot group, or whether the apparent association of mosaic hotspot mutations with PPB types I/IR is real. Consider: The hotspot mosaic children presented earlier in life for medical care. Mean age at diagnosis was less than one year. Early presentation and treatment limited time for disease progression. For other categories, there was almost certainly an ascertainment bias toward more aggressive PPB types. Many patients accrued to this study came to the attention of the PPB Registry through appeals from clinicians for advice on treating advanced PPB. Among germline LOF mutation carriers, the largest group of PPB patients by far, this typically means PPB already progressed to type II or III at the time of diagnosis. PPB type Ir (non-progressed) is likely under-diagnosed among germline mutation carriers. These are often single cystic lesions which, if small, may not cause breathing problems serious enough to elicit medical attention, and would be discovered only if thoracic imaging is done for another reason. What we can say with confidence is that mortality is higher among children presenting with PPB type II or III, as compared to PPB type I/Ir, regardless of genetic etiology. Minor Changes The authors should include appropriate references to any manuscripts in which any of these PPB registry cases have been previously reported. The reference list includes most previous publications concerning PPB registry patients in which any of the present authors participated. This study surveyed the medical records of 124 patients as well as information about most of their parents and numerous additional family members. Many of these individuals have been reported in multiple publications over the last twenty years or so, and not always with our participation. We will not undertake a comprehensive tabulation of all publications that mention any patient or family member included in this study. But if you have a specific interest in particular cases, please contact us directly, and we will do our best to help. While not essential, it would be informative to include any affected siblings for the 10 identified de novo LOF cases to support or refute potential germline mosaicism in the parents (Page 4)? There were no siblings or other family members identified with syndromic disease in the ten cases of de novo germline mutation. This information has been added to the text and revised supplemental Table S6. Clinical features of children with germline DICER1 LOF mutations. The de novo cases are those with a ‘0’ in the right-most column, indicating that both parents tested negative for the proband mutation. Table 1 includes a single case of a germline LOF mutation and a Wilms tumor (WT). However, little else is described about this case. Furthermore there is no mention of this case in the supplementary materials. Please include mutation analysis and additional phenotypic information for this case. It should have been indicated in Table S6 – thank you for catching the omission. The patient with a Wilms tumor is study ID# 108. The germline DICER1 mutation is c.1752+1delG, at the 5’ splice-site of intron 10. Skipping of exon 10 would cause in-frame deletion of 81 codons (V504 to K584) between the helicase and PAZ domains, the functional consequences of which are unknown. Actual effects of this mutation on splicing have not been examined. Syndromic disease in this male patient was confined to PPB type Ir and the Wilms tumor, which were diagnosed at age 36 months.The Wilms tumor was not available for sequencing, so we don’t know whether or what DICER1 mutations were present. The patient is living. Two family members had syndromic conditions, but have not been screened for mutation. This information is now included in revised supplemental Tables S4 and S6." } ] }, { "id": "10861", "date": "19 Oct 2015", "name": "James F. Amatruda", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, entitled “Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model”, Brenneman et al. present a comprehensive, state-of-the-art analysis of the various types of DICER1 mutations seen in patients with pleuropulmonary blastoma and other DICER1-related tumors. In their tumors, these patients often display a combination of a loss-of-function mutation on one allele of DICER1 and a hotspot RNase IIIb missense mutation on the opposite allele. As the authors show, tumors may acquire DICER1 mutations via germline inheritance, post-zygotic mosaicism, or somatic mutation at the time of tumor formation, and the mutations can be acquired in either order. Importantly, patients can be categorized based on the status of their inherited DICER1 alleles (wild-type, loss-of-function or hotspot missense; and germline or mosaic), and this categorization determines patient phenotype.\n\nThis is a timely report, given the recent surge of reports implicating DICER1 mutations in human cancers. The authors present a robust clinical and molecular characterization of a large cohort of PPB patients. The work is of high quality and the report is clearly written. The conclusions are largely supported by the data, with some possible exceptions outlined below. The large amount of supplementary data is a particularly valuable resource and serves as a model for studies of this kind. Overall this is a very valuable contribution to the literature on DICER1-related cancers. The reservations noted center a few remaining areas of ambiguity concerning the molecular model presented by the authors.Title“Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model”: This study does not appear to investigate DICER1 syndrome per se, but rather PPB. Inclusion criteria was “PPB patients (n=124)”, and this includes 12 patients who were found to have no germline or mosaic DICER1 mutation (Table S8) and another 12 patients who had no detectable DICER1 mutations (Table S9).IntroductionPPB is pathognomonic for a childhood cancer syndrome that features a range of other benign and malignant neoplasms including ovarian Sertoli-Leydig cell tumor (SLCT), …” As many of the DICER1-related cases of SLCT that have been reported occur in patients in their 30s and 40s (See for example Rio Frio et al., 2011), it is probable that DICER1 syndrome is not simply a “childhood” cancer syndrome. “Understanding how the interplay of RNase IIIb missense and LOF mutations influences the expression of syndromic neoplasias can aid diagnosis at early stages, when they are most curable.” As far as we know there are no data that early diagnosis of DICER1-syndrome neoplasias (other than, potentially, PPB) is beneficial. In fact, in this manuscript, the patients with mosaic hotspot mutations presented with lower-type PPB (non-sarcomatous) but underwent much more “complicated and arduous” clinical courses. “We propose that the extreme phenotypes of this patient group are attributable to the order in which allelic DICER1 mutations were acquired during development, i.e., an RNase IIIb hotspot missense mutation acquired early in embryogenesis and subsequently unmasked by LOF mutations or loss of the second allele.” The authors’ model (biallelic mutations are fundamental to the development of DICER1-related tumors, hence the need for a loss/LOF mutation in trans to a hotspot missense mutation) may not be universally true, or may be true for PPB but not other DICER1-realted tumors. While the frequency of biallelic DICER1 mutations appears to be high in PPB (this study; Pugh et al [Ref.6]; Seki et al [Ref. 35]), this does not appear to be the case in all DICER1-realted tumors. We previously reported a Wilms tumor patient with a DICER1 hotspot missense mutation and no detected germline mutation, though we did not rule out copy-number loss of the wildype allele (Rakheja et al., 2014). And the TARGET sequencing project reported DICER1 variants in Wilms tumor patients, including two patients who demonstrated non-hotspot germline missense mutations but no mutations on the other allele in their tumors (Walz et al., 2015).Methods“Definition of ‘disease foci’”: Several of the “disease foci” in Table 1 are left off this list (e.g., Wilms tumor, juvenile polyps of small intestine, ciliary body medullopeithelioma). Was this list dynamically expanded during the course of the study?ResultsWhat is the SIFT score for I582T? “no conclusive evidence of the variants in parental blood”: What was considered “conclusive evidence”? What read depth was obtained in these parents? “Preliminary data from an ongoing NCI-sponsored DICER1 family history study”: Understanding that these are unpublished data, it would be helpful if the authors could state approximately how large is the sample size. Do all asymptomatic patients in this study undergo thyroid ultrasound and lung CT? “We categorized a DICER1 mutation detected by NGS as mosaic when the following criteria were met: i. The mutation was evidently not a constitutional, germline allele because it was present at sub-heterozygous frequency (arbitrarily taken as below 35% of reads)”: According to Table S7, even in the two cases where tumor purity was 80-90%, % of reads supporting the loss-of-function allele was only 21%. This argues that either 35% is too high of a cutoff for determining subtotal mosaicism, or the tumors do not possess this mutation in every cell. Could the authors speculate on which is more likely? Table 2: The cystic nephroma in patient 103 and the Wilms tumor in Klein Case 1 both feature missense mutations in the non-hotspot allele (p.V377I and p.P453L, respectively, and the effect if any of these mutations on DICER1 activity is not known. Thus it may be premature to label these cases truly two-hit in nature. The fact that some tumors in children with mosaic hotspot mutations acquire several different LOF mutations (such as Study ID 103) agrees with the model. However the allele frequencies are overall low (3-4% in this tumor) making it unclear how significant the LOF event actually was. The authors may also want to consider the possibility that, in cells with a hotspot mutation and an intact WT DICER1 allele, 5p/3p miRNA skewing leads to defects in DNA replication or repair, predisposing to a second hit. Such a mechanism could in theory help explain the higher incidence of tumors in this group of patients, along with the fact that many more codons of DICER1 may be mutated to casue a LOF allele, compared with the hotspot missense mutations. Table 2: how was “allele loss” determined? Figure 3 is confusing, as pt 105 appears to be on a different x-axis than the other four patientsDiscussion“Additionally, we hypothesize that in mosaic hotspot children, wider tissue/organ distribution of aberrant miRNA processing during development may produce syndromic features not seen in children with predisposing LOF mutations, such as juvenile-type small intestinal polyps”: Could the authors speculate on why hotspot mutations would cause small intestinal polyps but LOF mutations would not? In the one case (#102, table 2) in which a polyp was sequenced, it was found to harbor both a LOF and hotspot mutation. As the hotspot mutation is the rate-limiting step, it seems more likely that LOF children also develop polyps, but at a lower frequency, and that their small intestines have not been thoroughly examined for the presence of polyps. “It may be impossible to unequivocally rule out mosaicism, but techniques such as targeted resequencing by high depth NGS in multiple tissues can greatly improve diagnostic confidence”: Usually, it is clinically unfeasible to perform high-depth NGS in multiple tissues from a patient. In patients whose germline DICER1 sequencing is negative, it may be more helpful to use clinical proxies to identify patients at high risk for mosaicism, such as young age and multifocal disease.OtherThroughout the manuscript, there are a few instances where “RNAse IIIb” is used instead of “RNase IIIb”. The latter is more standard nomenclature.", "responses": [ { "c_id": "3328", "date": "12 Jan 2018", "name": "D. Ashley Hill", "role": "Author Response", "response": "Author Responses in Bold to Referee Report James F. Amatruda, Departments of Pediatrics, Molecular Biology and Internal Medicine, University of Texas Southwestern Medical Center, TX, USA Kenneth S. Chen, Department of Pediatrics, University of Texas Southwestern Medical Center, USA Approved with Reservations In this manuscript, entitled “Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model”, Brenneman et al. present a comprehensive, state-of-the-art analysis of the various types of DICER1 mutations seen in patients with pleuropulmonary blastoma and other DICER1-related tumors. In their tumors, these patients often display a combination of a loss-of-function mutation on one allele of DICER1 and a hotspot RNase IIIb missense mutation on the opposite allele. As the authors show, tumors may acquire DICER1 mutations via germline inheritance, post-zygotic mosaicism, or somatic mutation at the time of tumor formation, and the mutations can be acquired in either order. Importantly, patients can be categorized based on the status of their inherited DICER1 alleles (wild-type, loss-of-function or hotspot missense; and germline or mosaic), and this categorization determines patient phenotype. This is a timely report, given the recent surge of reports implicating DICER1 mutations in human cancers. The authors present a robust clinical and molecular characterization of a large cohort of PPB patients. The work is of high quality and the report is clearly written. The conclusions are largely supported by the data, with some possible exceptions outlined below. The large amount of supplementary data is a particularly valuable resource and serves as a model for studies of this kind. Overall this is a very valuable contribution to the literature on DICER1-related cancers. The reservations noted center on a few remaining areas of ambiguity concerning the molecular model presented by the authors. Title “Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in DICER1 syndrome: a unique variant of the two-hit tumor suppression model”: This study does not appear to investigate DICER1 syndrome per se, but rather PPB. Inclusion criteria was “PPB patients (n=124)”, and this includes 12 patients who were found to have no germline or mosaic DICER1 mutation (Table S8) and another 12 patients who had no detectable DICER1 mutations (Table S9). The title has been revised to reflect our study’s emphasis on PPB: Temporal order of RNase IIIb and loss-of-function mutations during development determines phenotype in pleuropulmonary blastoma / DICER1 syndrome: a unique variant of the two-hit tumor suppression model We strove to survey the spectrum of pediatric neoplasias now associated with DICER1 mutations, as reflected by the summary of diverse organ sites involved in Table 1. As you surmised, this project began as a prospective study of predisposing DICER1 mutations in PPB. Associations of other tumor types with DICER1 mutation and the concept of an overarching DICER1 syndrome emerged over the study’s course. In practice, PPB remained a good inclusion criterion for a study of DICER1 syndrome because nearly all PPB is associated with DICER1 mutation, though as it turns out, not always germline mutation. For other tumors in the DICER1 spectrum, such as SLCT, the association is less constant and for some, e.g., Wilms tumor, it is rare. Moreover, it appears that most pediatric patients who present with DICER1 spectrum neoplasias of any kind, and are then confirmed to carry a germline or mosaic DICER1 mutation, have some degree of lung involvement even where PPB or lung cysts were not the first finding. If starting the study today, we would include patients who present with other syndromic neoplasias and no lung involvement. We have encountered some. Unfortunately, cases of that kind were not accrued prospectively or with as much supporting data. The 12 PPB children previously listed in Table S8 (now Table S9 in the revised supplemental tables) were categorized as having biallellic DICER1 mutations specific to the tumor, but this is a laboratory distinction – a reflection of what we could detect, not necessarily the underlying biology. They may represent a more limited form of mosaicism, i.e., mutations that occurred late in prenatal development and have a correspondingly restricted tissue distribution – perhaps so restricted that it gave rise to only a single focus of PPB in one lung. The 12 cases previously listed in Table S9 (now Table 10) should not be regarded as cases of PPB with no detectable DICER1 mutations. They were unresolved cases – ones we could not catagorize at that time. We have since confirmed two cases, study ID# 123, as an RNase IIIb hotspot mosaic (please see Table 2 in the revised manuscript). The Foulkes lab has since published results for study ID#120, indicating this patient is also a RNase IIIb hotspot mosaic, and we have some data supporting this. In nine of the ten remaining cases, tumor specimens are unavailable or too poorly preserved to sequence. DICER1 mutations have not been excluded in the tumors of those nine patients. The presence of cryptic mosaic or germline DICER1 mutations has been not been excluded for any of the ten patients. As this was a prospective study, we think it best to account for all the accrued PPB patients in this report, rather than omit some because of unresolved DICER1 mutation status. Introduction 1. PPB is pathognomonic for a childhood cancer syndrome that features a range of other benign and malignant neoplasms including ovarian Sertoli-Leydig cell tumor (SLCT), …” As many of the DICER1-related cases of SLCT that have been reported occur in patients in their 30s and 40s (See for example Rio Frio et al., 2011), it is probable that DICER1 syndrome is not simply a “childhood” cancer syndrome. The first paragraph of the Introduction has been revised to mention occasional diagnoses in adults. With regard to Rio Frio 2011, “many” may be a little too strong. One of five SLCTs in DICER1 mutation carriers described in that report was diagnosed at 32 years. The other four were diagnosed at ages in the teens or not specified. Table 2 of Rio Frio 2011 summarizes previously published descriptions of familial SLCT patients including several older adults, but those are much earlier papers and DICER1 mutation wouldn’t have been looked for. Similarly, Shultz et al 2012 identified 8 patients with germline DICER1 mutations and SLCT or other ovarian sex cord stromal tumors. One patient was diagnosed at 32 years; the others at ages ranging from 2 to 18 years. The larger question of whether and to what extent germline DICER1 mutations might figure in new onsets of neoplastic disease in adulthood is of great interest, but answers may have to await longer follow-up in identified families. 2. “Understanding how the interplay of RNase IIIb missense and LOF mutations influences the expression of syndromic neoplasias can aid diagnosis at early stages, when they are most curable.” As far as we know there are no data that early diagnosis of DICER1-syndrome neoplasias (other than, potentially, PPB) is beneficial. In fact, in this manuscript, the patients with mosaic hotspot mutations presented with lower-type PPB (non-sarcomatous) but underwent much more “complicated and arduous” clinical courses. Certainly the degree of benefit from earlier diagnosis will vary among DICER1 syndromic tumor types, as some have greater potential for malignant progression than others. For PPB, the benefits of early diagnosis are unequivocal. But rather than embark on a detailed breakdown in the Introduction, we have removed the phrase “when they are most curable”. With regard to the mosaic hotspot patients in this cohort, two had type II PPB and one has since died of metastatic disease. It’s important to remember the hotspot mosaic patients were recognized as such only in retrospect, after the analysis described in this report. Their clinical course was difficult, despite earlier diagnosis with (mostly) lower-type PPB, because of additional tumors in multiple organ sites. We still have no way to prevent that. But going forward, it will at least be possible to recognize hotspot mosaicism when present in children with PPB and/or other DICER1 syndromic tumors, and to anticipate exceptionally high risk for additional neoplastic disease. This improves our ability to educate caregivers and primary-care physicians about what to expect and watch for. 3. “We propose that the extreme phenotypes of this patient group are attributable to the order in which allelic DICER1 mutations were acquired during development, i.e., an RNase IIIb hotspot missense mutation acquired early in embryogenesis and subsequently unmasked by LOF mutations or loss of the second allele.” The authors’ model (biallelic mutations are fundamental to the development of DICER1-related tumors, hence the need for a loss/LOF mutation in trans to a hotspot missense mutation) may not be universally true, or may be true for PPB but not other DICER1-related tumors. While the frequency of biallelic DICER1 mutations appears to be high in PPB (this study; Pugh et al [Ref.6]; Seki et al [Ref. 35]), this does not appear to be the case in all DICER1-realted tumors. We previously reported a Wilms tumor patient with a DICER1 hotspot missense mutation and no detected germline mutation, though we did not rule out copy-number loss of the wild type allele (Rakheja et al., 2014). The TARGET sequencing project also reported DICER1 variants in Wilms tumor patients, including two patients who demonstrated non-hotspot germline missense mutations but no mutations on the other allele in their tumors (Walz et al., 2015). The model we propose is an attempt to explain (in part) why children with DICER1 mutations differ so dramatically with respect to numbers of disease foci and organ sites involved, and particularly the role of mosaicism. We share your view that a hotspot-plus-LOF model may not apply to all DICER1-associated tumors in all organ sites, especially with regard to some of the rarer manifestations of DICER1 syndrome – Wilms tumor, non-epithelial ovarian tumors and pineoblastoma particularly. We have extended the Discussion to address this. Methods 1. “Definition of ‘disease foci’”: Several of the “disease foci” in Table 1 are left off this list (e.g., Wilms tumor, juvenile polyps of small intestine, ciliary body medullopeithelioma). Was this list dynamically expanded during the course of the study? An oversight – thank you. The description in Methods now includes all neoplasias shown in Table 1. Results 1. What is the SIFT score for I582T? The SIFT score for I582T was 0.0, compared to a median of 3.55 for other substitutions at the analogous position in related genes of 11 species. It was classified as deleterious but of unknown pathogenicity. We have expanded Table S4 to show SIFT and PROVEAN results for all of the five germline (non-hotspot) missense mutations we found. 2. “no conclusive evidence of the variants in parental blood” What was considered conclusive evidence? What read depth was obtained in these parents? The putative de novo mutations in question were all single nucleotide variants, which can also occur as sequencing errors. In sequencing data from parental blood, “mutant” reads occuring at frequencies close to the predicted error frequency of the Ion Torrent platform were not taken as conclusive evidence of parental mosaicism. A new supplemental table, Table S5, summarizes the parental sequencing results, including read depths. 3. “Preliminary data from an ongoing NCI-sponsored DICER1 family history study”: Understanding that these are unpublished data, it would be helpful if the authors could state approximately how large is the sample size. Do all asymptomatic patients in this study undergo thyroid ultrasound and lung CT? Thyroid ultrasound and lung CT are offered to all identified DICER1 mutation carriers, but perhaps not all accept. Of 48 asymptomatic carriers so far screened by CT, 12 have lung cysts. The part of the study concerned with thyroid disease would be harder to summarize here, but has now been published: Khan et al, J. Clin. Endocrinol. Metab. 2017 May 1;102(5):1614-1622. doi: 10.1210/jc.2016-2954. PMID: 28323992 4. “We categorized a DICER1 mutation detected by NGS as mosaic when the following criteria were met: i. The mutation was evidently not a constitutional, germline allele because it was present at sub-heterozygous frequency (arbitrarily taken as below 35% of reads)”: According to Table S7, even in the two cases where tumor purity was 80-90%, the percentage of reads supporting the loss-of-function allele was only 21%. This argues that either 35% is too high of a cutoff for determining subtotal mosaicism, or the tumors do not possess this mutation in every cell. Could the authors speculate on which is more likely? That part of the sentence has been corrected to read “... because it was present at sub-heterozygous frequency in blood or normal tissue specimens (arbitrarily taken as below 35% of reads)”. No cutoff was applied for tumor specimens. But your point about allele frequencies in tumors of LOF mosaic patients is still valid. In the PPB tumor specimens of patients 89 and 90, allele frequencies of the detected LOF mutations are only about half of that expected for specimens having 80-90% tumor purity (revised supplemental Table S8). Even with allowance for inexact visual estimates of tumor purity, the conclusion seems inescapable: the detected LOF mutation is not present in every tumor cell. There is a similar instance in hotspot mosaic patient 111 (please see revised Table S9). Our working hypothesis is that the “missing” LOF alleles in these tumors are exactly that – deleted. For patient 111, an informative SNP marker linked to the DICER1 locus was also genotyped and the results support allele loss in the tumor. Moreover, there is evidence of allele loss in seven of the twelve sporadic PPB specimens in revised Table S9, which suggests some level of genome instability prevails in these tumors. 5. Table 2: The cystic nephroma in patient 103 and the Wilms tumor in Klein Case 1 both feature missense mutations in the non-hotspot allele (p.V377I and p.P453L, respectively) and the effect if any of these mutations on DICER1 activity is not known. Thus it may be premature to label these cases truly two-hit in nature. Yes, it may be. The only other mutations in our data set resembling these two are the I582T germline mutation in patient 36 (noted in item 1, above) and the L278F mutation in patient 107 (detected in a PPB tumor and at very low level in blood). Like V377I and P453L, these missense mutations are in or near the N-terminal helicase domain of DICER1, the functional importance of which is uncertain. An additional missense mutation, R746G, lying between the helicase and PAZ domains, was found at low allele frequency in the blood of patient 92 (revised Table S8). This was classified as a mosaic LOF allele solely on the basis of its presence in a child with PPB (type III; unfortunately not available for sequencing). To assist readers in coming to their own conclusions, additional annotation is provided for each of the missense mutations in revised Table S4. 6. The fact that some tumors in children with mosaic hotspot mutations acquire several different LOF mutations (such as Study ID 103) agrees with the model. However the allele frequencies are overall low (3-4% in this case), making it unclear how significant the LOF event actually was. Yes, if we assume there was only the detected LOF event and no other. The confounding problem is that we have not ruled out cryptic LOF mutations or allele losses not readily detected by the methods we used. It’s fairly straightforward to show that a nonsense or frameshift mutation is present or not, as they must occur in coding sequence. But cryptic LOF mutations could be any kind of event from single nucleotide changes in non-coding regulatory sequences to chromosomal rearrangements that preserve all wild type coding sequence but de-link the exons. Allele loss can be difficult to establish on the basis of read counts in specimens of low tumor purity. 7. The authors may also want to consider the possibility that, in cells with a hotspot mutation and an intact WT DICER1 allele, 5p/3p miRNA skewing leads to defects in DNA replication or repair, predisposing to a second hit. Such a mechanism could in theory help explain the higher incidence of tumors in this group of patients, along with the fact that many more codons of DICER1 may be mutated to cause an LOF allele, compared with the hotspot missense mutations. We’re very interested in that possibility too, but chose not to speculate, because we had no data showing 5p/3p miRNA skewing in tumors or tissues with one hotspot allele and one wild type. The best and only data of that kind we know about is your own. In Rakheja et al 2014, Figure 3d shows clear skewing of 5p/3p miRNA output from Wilms tumor specimen CMCW59, in which only a G1809V hotspot mutation was detected. Although skewing is not so extreme as in CMCW11 (G1809R + frameshift LOF), it still amounts to inversion of the characteristic 5p/3p ratio seen in wild type specimens, and it’s hard to imagine this has no consequence for gene regulation. A related possibility is that DICER1 RNase IIIb hotspot mutations exert a dominant negative effect over wild type DICER1, as you demonstrated for RNase IIIb mutation in DROSHA. What the mechanism of a dominant negative effect on miRNA processing by mutant DICER1 might be is unclear. A difficulty of interpretation for tumors like CMCW59 or tumors of patients 120, 123 and 124 in this study, in which only a hotspot muatation has been detected, is to rule out cryptic LOF mutation. It’s hard to be certain whether a tumor’s genotype is truly DICER1hotspot/wildtype or actually DICER1hotspot/LOF. Perhaps the best way to model effects of a DICER1hotspot/wildtype genotype on miRNA processing and gene expression would be a genome editing approach like that used in Rakheja et al 2014 for DROSHA. Effects of DICER1 hotspot mutations on genome stability might well arise through 5p/3p miRNA skewing. Over thirty genes related to DNA repair or replication have shown altered expression levels in PPB tumors (Hill et al, unpublished). But in thinking about genome stability and mutagenesis, we also wonder whether catalytic activities of DICER1 (or DROSHA) might figure in other nucleic acid transactions besides miRNA processing. Could they have more direct roles in replication or repair? 8. Table 2: how was “allele loss” determined? Allele loss was inferred from allele frequency disparities. In eight separate tumors arising in four hotspot mosaic patients (102, 104, 105 and 123), no specific LOF coding sequence change was detected and relative frequency of the hotspot missense allele is equal to or greater than the estimated tumor purity of the specimen. This implies that all or nearly all copies of DICER1 present in tumor cells are the hotspot missense allele, i.e., the second allele has been lost in most tumor cells. In the example calculations that follow, “wild type” means only that no mutation was present in amplicons representing exons 24 or 25, where the hotspot codons are located; an LOF mutation could be present elsewhere in the gene. Example 1. In the left ovarian SLCT of patient 104, an estimated 95% of cells in the specimen are tumor cells and we assume they all carry the hotspot missense allele. Let 2n be the total number of DICER1 alleles present in a sample of tissue or tumor comprising n diploid cells. Consider three hypothetical allele distributions: A. If both a hotspot missense mutant allele and a wild type allele are present in all tumor cells, and two wild type alleles are present in non-tumor cells making up the remaining 5% of the specimen, then: Hotspot missense allele frequency would be: (.95 x 1n) / 2n = 47.5% Wild type allele frequency would be: (.95 x 1n + .05 x 2n) / 2n = 1.05n / 2n = 52.5% B. However, if all tumor cells are hemizygous at DICER1, retaining only the hotspot missense allele, and all non-tumor cells are homozygous wild type, then the total number of DICER1 alleles present in the specimen would be reduced from 2n to only 1.05n, i.e., 0.95 x 1n + 0.05 x 2n. Hotspot missense allele frequency would then be (.95 x 1n) / 1.05n = 90.5% Wild type allele frequency would be (.05 x 2n) / 1.05n = 9.5% C. Alternately, if all tumor cells are hemizygous but the 5% non-tumor cells are all heterozygous (one missense allele and one wild type), then: Hotspot missense allele frequency would be (.95 x 1n + .05 x 1n) / 1.05n = 95.2% Wild type allele frequency would be (.05 x 1n) / 1.05n = 4.8%. The actual allele frequencies measured in this specimen were 92.4% hotspot missense and 7.6% wild type. These frequencies are intermediate to those predicted in alternatives B and C above for tumor cell populations hemizygous at DICER1 (having lost their wild-type allele). The ~ 5% non-tumor cells in this specimen might be accounted for as a mix of heterozygous (hotspot mutant /wild type) and homozygous wild type cells, consistent with somatic mosaicism. Example 2. Patient 102, PPB brain metastasis: estimated tumor purity 30% A. If both a hotspot missense mutant allele and a wild type allele is present in all tumor cells, and two wild type alleles are present in non-tumor cells making up the remaining 70% of the specimen, then: Hotspot missense allele frequency would be: (.30 x 1n) / 2n = .30n / 2n = 15% Wild type allele frequency would be: (.30 x 1n + .70 x 2n) / 2n = 1.7n / 2n = 85% B. If all tumor cells are hemizygous at DICER1 (having lost their wild-type allele) and all non-tumor cells are homozygous wild type, then the total number of DICER1 alleles present in the specimen would be reduced from 2n to (0.30 x 1n) + (0.70 x 2n) = 1.70n, and: Hotspot missense allele frequency would then be (.30 x 1n) / 1.7n = 17.6% Wild type allele frequency would be (.70 x 2n) / 1.7n = 82.4% C. Alternately, if all of the 30% tumor cells are hemizygous but the 70% non-tumor cells are all heterozygous (one missense allele and one wild type), then: Hotspot missense allele frequency would be (.30 x 1n + .70 x 1n) / 1.70n = 58.8% Wild type allele frequency would be (.70 x 1n) / 1.70n = 41.2%. The measured frequencies for this specimen were 51% hotspot mutation and 49% wild type. This appears most consistent with alternative C. Again, the non-tumor cells may be a mix of heterozygous and homozygous wild type cells, making the observed hotspot allele frequency somewhat lower than predicted and the wild type correspondingly higher. Although other explanations for the allele frequencies seen in these tumors are certainly conceivable, allele loss appears the most straightforward. 9. Figure 3 is confusing, as patient 105 appears to be on a different x-axis than the other four patients. In Figure 3, the clinical time line (x-axis) of patient 105 is interrupted at 7½ years (double slash symbol) and compressed to the right of that point so that events out to 17 years could be shown. This is a common graphical device, but admittedly less than ideal. We could alternatively show all patients on a longer, unbroken time scale, but that would require compressing the entire x-axis to fit the figure on a page. On balance, we thought it best to keep the interrupted timeline for patient 105 and preserve detail in the earlier years for all patients. Discussion 1. “Additionally, we hypothesize that in mosaic hotspot children, wider tissue/organ distribution of aberrant miRNA processing during development may produce syndromic features not seen in children with predisposing LOF mutations, such as juvenile-type small intestinal polyps”: Could the authors speculate on why hotspot mutations would cause small intestinal polyps but LOF mutations would not? In the one case (#102, Table 2) in which a polyp was sequenced, it was found to harbor both a LOF and hotspot mutation. As the hotspot mutation is the rate-limiting step, it seems more likely that LOF children also develop polyps, but at a lower frequency, and that their small intestines have not been thoroughly examined for the presence of polyps. All the instances of juvenile intestinal polyps in mosaic hotspot children were discovered at surgery for intestinal intussusception. It may be that some children with germline or mosaic LOF mutations also developed juvenile intestinal polyps. If less numerous or smaller and not associated with intussusception, those might well escape notice, because examination of the small intestine for polyps has not been usual in diagnostic workup of children presenting with PPB or other DICER1 syndromic tumors. But we were alluding to the possibility you suggest in item 7 (Results) above – that gene regulation is materially altered in cells bearing a mosaic hotspot mutation, even while a wild type allele is retained. In those embryonic tissues where a large fraction of stem or progenitor cells are DICER1hotspot/wildtype, some degree of 5p/3p skewing could conceivably have effects such as delayed cell lineage committment or incomplete differentiation, resulting in developmental disturbances, e.g., exaggerated growth of localized tissue regions or entire organs. 2. “It may be impossible to unequivocally rule out mosaicism, but techniques such as targeted resequencing by high depth NGS in multiple tissues can greatly improve diagnostic confidence”: Usually, it is clinically unfeasible to perform high-depth NGS in multiple tissues from a patient. In patients whose germline DICER1 sequencing is negative, it may be more helpful to use clinical proxies to identify patients at high risk for mosaicism, such as young age and multifocal disease. We were thinking of normal adjacent tissue recovered at tumor resection, together with sources that can be sampled without surgical biopsy, such as blood and buccal cells. In the future, development of digital droplet PCR assays for the limited spectrum of known RNase III hotspot mutations might eliminate any need for deep sequencing. But your point on clinical proxies is a good one and we have revised the paragraph. In a child who presents very early with multifocal disease, hotspot mosaicism should be suspected even if specimens for high-depth NGS are not available. More problematic is the child who presents early with a single, DICER1-mutant tumor and tests negative for germline mutation. Is this child at risk for additional tumors or not? Other 1. Throughout the manuscript, there are a few instances where “RNAse IIIb” is used instead of “RNase IIIb”. The latter is more standard nomenclature. Corrected – thank you." } ] } ]	1	https://f1000research.com/articles/4-214
https://f1000research.com/articles/7-47/v1	11 Jan 18	{ "type": "Research Article", "title": "Global Open Data in Agriculture and Nutrition (GODAN) initiative partner network analysis", "authors": [ "Ruthie Musker", "Ben Schaap" ], "abstract": "Background: Ensuring healthy, safe and nutritious food for everyone is a global concern. Accessing the information to make the correct decisions regarding food security can be challenging. Open data has been shown to help solve practical problems related to agriculture and nutrition, enabling effective decision-making. In order to create a global data ecosystem that benefits everyone, a wide range of stakeholders must be included in the conversations. The GODAN initiative involves a network of over 500 partner organizations committed to open data in agriculture and nutrition. Methods: We analysed data from a survey of the partner organizations, with 225 respondents, to determine open data activities, including challenges, use of open data, stakeholder involvement and future directions. Respondents were asked a variety of free text and multiple choice questions. Results: 160 partners had at least one open data activity, 65 did not, or did not know. Of the 160, 36 had a second activity. Overall, GODAN partners are developing 200 open data activities. Agriculture is the most common focus for an open data activity. Nutrition-only activities are strongly underrepresented. The most frequently mentioned challenge was cost, which is linked to data governance, management, and human capacity; many do not have the funding to begin or maintain open data activities. Conclusions: The most common challenges were the ones related to the data itself, including how to access it, manage it, and how to keep the sensitive data secure. GODAN is already focusing on these issues through the Responsible Data and Data Ownership pieces. Capacity building, and empowering partners with the tools they need to act, is one of the most effective actions available for GODAN. Funding for open data, as well as research to create more sustainable business models, should be the focus of the open data agenda.", "keywords": [ "open data", "agriculture", "nutrition", "collaboration", "partnerships", "godan" ], "content": "Introduction\n\nThe challenge of global food security is expected to intensify over the coming decades due to an increase of 2 billion people on the planet by 2050 and 1 billion people at risk of hunger and malnutrition in the same time frame1,2. Food security can hopefully be achieved through sustainable agriculture, innovative business models, and political will, however, access to information will be crucial to achieve this goal. Open data, which is data anyone can access, use, or share3 is key for access to information, and research has shown that open data can help enable effective decision-making and practical problem solving. Open data and transparent processes can trigger organization and sector change to provide innovations to benefit all3. The Global Open Data for Agriculture and Nutrition (GODAN) initiative uses the FAIR principles (Findable, Accessible, Interoperable, and Reusable) to conceptualize the full meaning of open data4. Open data does not exist in isolation, it must develop in the context of a global data ecosystem, which considers all stakeholders and their data needs5.\n\nA vast amount of data and information has been gathered about agriculture, food security, and nutrition, which vary by language, units, size, subject matter, and management structure and process. Agriculture alone has an especially large number of stakeholders involved, all of which are in multiple locations within the agricultural supply chain (provider to consumer of crops) and the data supply chain (provider to consumer of data). Partnership, collaboration, and data sharing are important for two stages:\n\n1) To create a global data ecosystem that is useful for all stakeholders, while releasing data responsibly, and with consideration of data ownership and security; and\n\n2) To use the global data ecosystem to achieve global food security5.\n\nOpen data in agriculture and nutrition is an emerging topic, and often a delicate one. Individuals and organizations are not sure of best practices, challenges, and consequences of releasing data openly. Cooperation, collaboration, and partnerships are necessary to building trust in creating a shared data ecosystem. The GODAN Partner Network (currently in November 2017) has 600 partners from national governments, non-governmental, international and private sector organisations that have committed to a joint Statement of Purpose6, provides a collaborative space to convene like-minded people who seek to advance open data in the agriculture and nutrition agenda.\n\nWhen an organisation commits to join GODAN partner network, they agree to:\n\nadvocate for open data initiatives for agriculture and nutrition\n\nrelease agriculture and nutrition data\n\nincrease awareness of agriculture and nutrition open data initiatives\n\nadvocate for collaboration amongst the partnership network\n\nadvocate for good practices and lessons learned for open data in agriculture and nutrition\n\nThis research article paper analyses their activities and challenges, to learn how GODAN can equip partners with tools they need, empower them to achieve their goals and overcome challenges, and convene partners together both in workshops and online to build trust and collaborative power. Additionally, this information can help others learn from the GODAN partner network and to build on the research.\n\n\nAbout GODAN\n\nThe Global Open Data for Agriculture and Nutrition (GODAN) initiative was the result of the 2012 G-8 Summit and the 2013 G8 International Conference on Open Data for Agriculture. The initiative and Secretariat was formally announced at the Open Government Partnership Conference in October 2013.\n\nG-8 leaders created GODAN to “share relevant agricultural data available from G-8 countries with African partners” and to ‘obtain commitment and action from nations and relevant stakeholders to promote policies and invest in projects that open access to publicly funded global agriculturally relevant data streams, making such data readily accessible to users in Africa and world-wide, and ultimately supporting a sustainable increase in food security in developed and developing countries.’\n\n\nMethods\n\nIn order to become a GODAN partner, organizations register on the GODAN website (www.godan.info/partners), which is linked to a Customer Relationship Management (CRM) system (CiviCRM). When registering, potential partners must give organization name, freetext info, location, website, logo, organization type, and point of contact information. This is the only information GODAN has on all partners. Partners must then agree to a commitment to open data in agriculture and nutrition, or recognize the importance of open data to achieve food security goals. There is no fee or membership cost. The reason for this is to allow the partner network to be as inclusive as possible. Once a partner has registered, a point of contact from the organization is sent a link to the survey. Partners can choose to take the survey or not. GODAN partners that have responded to the survey are hereafter referred to as “respondents”.\n\nThe purpose of GODAN is two-fold: to help raise awareness about the different open data activities that are happening in the network, and to help organizations who are seeking data to find potential partners and overcome open data challenges. At its inception, the GODAN Secretariat believed that in order to facilitate partnerships and effectively advocate for open data, the Secretariat must survey partners and their open data activities. The survey was created in Survey Monkey collaboratively by eight members of the GODAN Secretariat. The complete survey “BQ sample”, as it was presented to GODAN partners, is included as a PDF document in this research article (Supplementary File 1). The survey aims to profile the organizations and their open data activities, as well as the challenges that accompany these challenges.\n\nRespondents are asked a variety of both free text and multiple choice questions to clearly state their opinions, challenges, and needs in open data, and the various open data activities they are involved with. This information helps both partners and the wider community to see how open data in agriculture and nutrition is developing, and exactly who is doing what. Respondents do not have to complete the entire questionnaire. The first GODAN partner survey was sent to partners on April 10, 2015 and was sent to partners as they joined. On February 10, 2017, the GODAN Secretariat revised the survey. This paper analyzes the results of the first survey.\n\nBoth authors of this article analysed the results of the survey using Microsoft Excel 2013 downloaded as a CSV file from Survey Monkey. An anonymised version of this downloaded CSV file is included as a dataset in this article (Dataset 1), which does not include name of organization or contact info). To ensure privacy, we removed the information about challenges, and included it as a separate spreadsheet (Dataset 2). Quantitative results used formula analysis in Excel, and qualitative results involved Excel search functions and human confirmation of results.\n\n\nEthical statement\n\nConsidering the absence of identifying information in data published in aggregated form here, and the non-sensitive nature of the survey, no ethical approval was sought for this study. No information presented here can be used to identify survey participants, and in accordance with SurveyMonkey’s data privacy policy (https://www.surveymonkey.com/mp/policy/privacy-policy/), is not accessible to third parties.\n\nIt is not a requirement of the GODAN partner network to fill out the survey. When sending the survey to new partners, we state, “After analysing the questionnaire data, we will use the aggregated information to show how the open data community in agriculture and nutrition is developing and who is doing what”, thus resulting in this research article.\n\n\nResults\n\nBetween April 10, 2015 and February 6, 2017, 225 of 432 GODAN partners had filled out the partner survey. Nine were from different representatives of the same organization. This represents 53% of the GODAN Partner base at that time. Geographical and sector data is collected from partners at registration (Table 1, Table 2). Generally, the survey is a balanced representation of our partnership network as a whole. However, our partnership network is heavily skewed towards Africa, Europe and North America. The survey is also skewed towards universities and research institutions, and private sector completing the survey compared to its distribution in the Partner Network.\n\nGODAN partners primarily use open data to achieve goals around sustainable food production and food security (Table 3). The focus of open data is primarily on economic gain, with the social aspects less of a focus, especially gender balance.\n\nMultiple choice question, respondents could choose more than one option. The numbers represent how many times the options were selected.\n\nWhen the survey began, the GODAN Secretariat believed that those who joined GODAN would already be working with and producing open data and have open data activities to share (Table 4).\n\nMultiple choice question.\n\n160 partners have at least one open data activity, 65 do not, or do not know. Of the 160, 36 have a second activity, and 4 have a third. Overall, GODAN partners are developing 200 open data activities. Agriculture is the most common focus for an open data activity, with a joint agriculture and nutrition activity close second. Nutrition-only activities are strongly underrepresented (Table 5). A general observation is that most of the “neither” responses focus generally on open data, open access and open government (see Anonymized Partner Survey Spreadsheet).\n\nMultiple choice, respondents selected agriculture and/or nutrition, ‘neither’ was when the respondent did not check either agriculture or nutrition.\n\nMost GODAN partner respondents are involved with data collection and publishing (Table 6). However, about half of respondents were involved with four or more aspects of open data listed in Table 6. Sixteen respondents were involved with all seven aspects, 26 with six, 38 with five, and 37 with four.\n\nRespondents were allowed to choose more than one option.\n\nData collection means sourcing any data directly through research, instrumentation, surveys or other methods. Publishing includes producing static products drawing upon your own open data, and/or open data from others. A data intermediary makes open data more accessible for others, through creating applications, interfaces or derived datasets. A service provider uses open data to support services such as farm extension, weather information, market information, etc. A data provider makes open data available to others, and an end user uses open data directly, or through an intermediary, to affect their practice (e.g. farmer/farmers’ organisation, advocacy organisation, practitioners). These details were given along with the question (see BQ sample; Supplementary File 1).\n\nTable 7 analyses activities the respondents described. All partners were asked an open-ended question to describe their open data activity with no text limit. To analyse the activities, the GODAN research team created a word frequency table (Table 7) based on text mining in the free text sections and categorized them. When text mining, the authors used the search method in Excel. Activities marked with an asterisk () show the search term used, which accounts for variability in the ending of the word (ex: app includes app, apps, application, applications). The authors personally viewed the results to ensure that the word was used in the correct context. (ex: “approach” is not included under app*). The words were checked by one of the authors to ensure that they were taken in the correct context. All information to determine these results are in Anonymized Partner Survey Spreadsheet.\n\nQuestion was freetext.\n\nUnder methods of working, collaboration, sharing, and open access are on the top of the list. In terms of outputs, research and publications were the highest (but we are not sure if it is research data and published data and otherwise), and platforms, portals, and tools feature highly as well. Some initiatives, centers, and policies are created as well.\n\nGovernments are the most common stakeholder to engage with when it comes to open data, which makes sense since they are both large users and producers of data, and have the capacity to gather data. Researchers and farmers come next, which also makes sense since researchers are primarily looking for data to complete their research, while farmer’s data is valuable to almost all within the food system. (Table 8).\n\nMultiple choice question, respondents could choose more than one option.\n\nThe majority of respondents are engaged with more than one stakeholder group, and many engage with 2–4. Several engage with more, but only one engages with all stakeholders (Table 9).\n\nMultiple choice question, respondents could choose more than one option.\n\nRespondents were asked the open-ended question: “What are the key challenges your organisation faces in developing this activity further with respect to open data? Please share your insights in a few sentences.” Respondents were encouraged to answer in their own words. Each response was individually read and analysed by a member of the GODAN Secretariat Research team. Through this process, the answers were aggregated into a format we could analyse (Table 10). For example, those who mentioned “financial issues”, “needing funding”, or “monetary burden” in their challenge were placed into “cost” category. The challenges are categorized by buy-in, data, resources and skills, methods, culture and other. These challenges stem from the activities listed above and can be found in the Challenges Partner Survey Spreadsheet”.\n\nThe most frequently mentioned challenge was cost; many do not have the funding to begin an open data activity or to maintain one. It costs money to train in open data management and, often, people with those skills are more expensive to employ. Managing and accessing open data is difficult as well, even if cost isn’t an issue. Convincing specific sectors of the importance of open data and actually buy-in to the open data agenda is a big challenge as well.\n\n\nConclusions\n\nThe partner survey has become a strategic resource for the Secretariat to develop the GODAN initiative towards; 1- geographic, topical focus and stakeholder group representation and 2 - stakeholder needs in terms of support (capacity building, providing resources, building advocacy). The GODAN initiative and its partners provide inspiration, show best practices and connect with partners beyond the current network. The purpose of the GODAN Secretariat is to facilitate partnerships and provide our partnership network with resources to advance the open data agenda and this publication is one of those resources.\n\nThe survey has a balanced representation by region of those who have answered the survey and our partnership network as a whole. However, our partnership network is heavily skewed towards Africa, Europe and North America. GODAN hopes to improve our partnership network representation globally to have equal representation and holistic understanding of the state of open data activities in agriculture and nutrition. In order to do this, the GODAN Secretariat could adjust our advocacy messaging to more specific regional audiences.\n\nThe survey is skewed towards universities and research institutions completing the survey compared to its distribution in the Partner Network. This also makes sense as to why research is a primary activity output (as listed in Table 7). Increased government input to the survey would be tremendously useful, especially since lack of policy and lack of government buy-in is a significantly mentioned challenge and a number of our partners express the need for assistance in convincing governments why open data is important. Together with Open Data Charter, GODAN is in the process of developing an Agriculture Open Data Package for governments7 to help with this goal.\n\nThe use of data in gender equality is very much underrepresented in the GODAN partner network. While 42 respondents stated they are working on gender balance, it wasn't clear if any respondents open data activities focused on gender, neither as gender data used or provided, nor empowerment of women and girls. Since researchers have concluded that an equal gender balance is essential for positive sustainable agricultural and nutrition outcomes8, the absence of any gender-focused activities is unfortunate. We must focus on not only improving our gender data representation within GODAN, but also emphasizing the message that partners must integrate gender considerations into their work.\n\nAlthough GODAN aims to focus on agriculture and nutrition, the number of nutrition specific focused initiatives is low. However, a large number of activities focus on both agriculture and nutrition which is a link that spans the supply chain and connects various sectors. When we consider the data supply chain, and the movement of a certain data point as it provides information from one stakeholder to the next, we have a large number of activities that can help facilitate this work.\n\nBased on the results of Table 7, a large number of partners focus on applications of data instead of data infrastructure, and lack of data infrastructure is a challenge. The need for data to create applications may drive the development of data infrastructure, however, data infrastructure must develop alongside the applications as well so they can constantly inform each other. Interoperability is crucial especially when working at a global scale. Currently, existing data infrastructures seem to be in their infancy, as many pre-requisites such as common vocabularies, ontologies and exchange standards require a collaborative effort from the user community. While some sectors are developing fast (eg. genomics9,10, precision agriculture11) others are developing much slower or have not even started to think about the prerequisites for data infrastructures.\n\nThe most common challenges, collectively, are the ones actually related to data. How to find it, access it, manage it, store it, organize it, keep the sensitive data secure, and ensure that the rightful owners are ensured the benefits. GODAN is already focusing on these issues through our Responsible Data and Data Ownership pieces12,13, and various working groups14 on the subject. However, we realize that these challenges are not simple and will have many solutions according to the context. No respondents’ activity is focusing on data ownership, or FAIR data, however, through personal communication with partners who did not answer the survey, GODAN does know other partners, such as DTL and the Engine Room, are working with FAIR data.\n\nA common challenge was the culture of opening data and the shifting of mindset to integrate data stewardship into the workflow of each sector and prioritizing effective data governance. Many people are not aware of open data or and even if they do, they do not have a specific and trusted path to follow to implement good (open) data stewardship into their work. Funding schemes are changing and may even evolve further to take these components into account. And the way research is performed much of the outcomes need to realign to the needs of a global data ecosystem. The GODAN partner base could be leveraged to determine some solutions to these issues and find support for good data stewardship.\n\nCapacity building, and empowering partners with the tools they need to act, is one of the most effective actions that GODAN can do. The GODAN Capacity Building Working Group15 is focusing on training for open data advocacy, publishing of open data, and developing business models for open data.\n\nCost is the most listed challenge and is linked to data governance, management, and human capacity. Funding for open data work as well as research and practical solutions on sustainable business models for open data should be an essential component of the open data agenda.\n\nAt GODAN, we would like to utilize our partner base to engage with them as much as possible. Now that, through the survey, we understand our partner’s needs and challenges better, we can work, alongside other initiatives, to equip partners with tools they need, empower them to achieve their goals and overcome challenges, and convene partners together both in workshops and online. Our common results and output can help others to learn from the GODAN partner network, to build on our research, and help foster collaboration, trust, and innovation to combat world food insecurity, develop sustainable agriculture and provide safe nutritious food.\n\n\nData availability\n\nDataset 1: Anonymized Partner Survey Spreadsheet. Directly downloaded from Survey Monkey. DOI, 10.5256/f1000research.13044.d18952216.\n\nDataset 2: Challenges Partner Survey Spreadsheet. Directly downloaded from Survey Monkey. DOI, 10.5256/f1000research.13044.d18952317.", "appendix": "Competing interests\n\n\n\nBoth authors of the publication are employees of the GODAN Secretariat.\n\n\nGrant information\n\nGODAN and the GODAN Secretariat are funded by the United States Department of Agriculture (USDA), the UK Department for International Development (DFID), the Dutch Ministry of Economic Affairs, and the Food and Agriculture Organization of the United Nations (FAO).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: BQ sample, directly downloaded from Survey Monkey. Survey sent to participants, which was tested by an author.\n\nClick here to access the data.\n\n\nReferences\n\nEstimates, UN Population Division: World Population Prospects: The 2015 Revision. 2015. Reference Source\n\nIFPRI: Food Security in a World of Natural Resource Scarcity: The role of agriculture technologies. 2014; 250. Accessed 20/04/17. Reference Source\n\nOpen Data Institute: How can we improve agriculture, food and nutrition with open data? 2015. Reference Source\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. Publisher Full Text\n\nGODAN: A Global Data Ecosystem for Agriculture and Food. 2016. Reference Source\n\nGODAN: Statement of Purpose. 2016. Reference Source\n\nGODAN: Agriculture Open Data Package: Beta. 2016. Publisher Full Text\n\nQuisumbing AR, Meinzen-Dick R, Raney TL, et al.: Gender in agriculture: closing the knowledge gap. Springer Science & Business. 2014. Reference Source\n\nGODAN: Responsible Data in Agriculture. 2016. Reference Source\n\nBig Data Infrastructure for Crop Genomics. Reference Source\n\nHe KY, Ge D, He MM: Big Data Analytics for Genomic Medicine. Cho WC, ed. Int J Mol Sci. 2017; 18(2): pii: E412. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPrecision agriculture and the future of farming in Europe Scientific Foresight Study. Reference Source\n\nGODAN: Ownership of Open Data: Governance Options for Agriculture and Nutrition. 2016c. Reference Source\n\nGODAN Working Groups. Reference Source\n\nGODAN Capacity Building Working Group. Reference Source\n\nMusker R, Ben S: Dataset 1 in: Global Open Data in Agriculture and Nutrition (GODAN) Initiative Partner Network Analysis. F1000Research. 2018. Data Source\n\nMusker R, Ben S: Dataset 2 in: Global Open Data in Agriculture and Nutrition (GODAN) Initiative Partner Network Analysis. F1000Research. 2018. Data Source" }	[ { "id": "29721", "date": "30 Jan 2018", "name": "Karel Charvat", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nScientific paper \"Global Open Data in Agriculture and Nutrition (GODAN) Initiative partner network analysis” addresses important aspect of Open Data in Agriculture and Nutrient. It describes and analyses survey, which was provided by GODAN among its members. It give overview about collection and utilisation of Open Data by GODAN member. It give basic statistic of different types of GODAN members and also their role in Open Data Chain. From this point of view review is well provided.\nWhat is missing is to put this paper into broader content. In some parts it has more form of reports, then scientific paper. Analysis of literature is relatively poor and it is mainly focused on previous GODAN analysis without clear reference. There are also mistake in links from text to references (more in end of paper).\nThere exist number of research activities focused on Open Data for Agriculture and Nutrient, but the results of review are not compared with this research. Due near focus only on interpretation of survey, there are missed information about some global initiatives like for example GEOSS (which cooperate closely with GODAN) and which is partly focused on Open Data for Agriculture. What is also missing in survey is focus on type of data. This is important due the fact, that for example satellite data (Landsat, Sentinel) are now currently biggest source of Open Data for Agriculture, which are broadly used.\nStatistic are well elaborate, but in some cases samples are relatively poor and it could be also influenced, that samples are taken only from GODAN members. But on other side is clear, that it is not easy to collect such information.\nProbably will be also good to mentioned in article question of data privacy. It is partly addressed in Conclusion, but without clear explanation. It is clear that not all data could be open due privacy, but it is not mentioned clearly. In relation to this I would like also recommend to compare open data and shared data. This play important role for example in Precision Agriculture, where number of applications is based on combination of Open Data (for example satellite, and data, which are shared like private farming data).\nConclusion is analysing results of survey and presenting importance of analysis, for other GODAN activities. However is a little generic, it will be good to stress some important activities and source, which could be used for future for GODAN activities, and also how better target activities in certain regions.\nIn conclusion about data infrastructure (page 8) is not clear if discussion is about infrastructure for Open Data or generic Data Infrastructure. From the text seems, that more about generic data infrastructure.\n\nThere are in conclusion also wrong links to references. For example reference 11, which is in text related to Precision Agriculture is focused on previous Genomic. Then Reference 12 related to Precision Agriculture is mentioned in text in relation with Data Ownership (please check this). It is also not clear, if the reference 12 \"Precision agriculture and the future of farming in Europe Scientific Foresight Study\" is really relevant to the topic of Data Infrastructure, resp. Open Data Infrastructure.\nI would like recommend to modified this article and put it into broader content of Open Data for Agriculture and Nutrient and also clarify some not clear statement in Conclusions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "29722", "date": "13 Feb 2018", "name": "Joel Gurin", "expertise": [ "Reviewer Expertise Open data", "with focus on its use in development and for business applications" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study of open data and its application has relied largely on case studies with relatively little quantitative survey work and analysis. From that perspective, this GODAN survey is a step forward and a useful contribution to the literature. However, the authors' report seems to miss some important opportunities to analyze data that they appear to have gathered through their questionnaire.\nThe study design is methodologically sound. The survey instrument is straightforward and clear, and the response rate (over 50%) is good for this kind of survey in this field. While the authors spend some time discussing the differences between the distribution of respondents and the distribution of all GODAN partners - both regionally and by sector (government, private sector, etc.) - those differences in fact appear to be fairly minor (with the caveat that I am not a statistician). So the survey was well designed and got a good overall response that represented GODAN's constituent groups fairly.\nThat said, the analysis and conclusions are disappointing. The data presented are largely descriptive of GODAN partners' activities, without shedding much light on how GODAN can better help these partners achieve their goals, which is the stated purpose of the study. There are some interesting findings, which the authors note: An apparent lack of attention to gender and nutrition per se, which may show a need for better strategic communication on these issues, and the clear indication that cost is a barrier to instituting open data programs. (This last may seem obvious, but the cost of open data programs has been a subject of some debate in the community working to apply open data for development.) However, most of the analyses presented are unsurprising and not as illuminating as they could be.\nSome further work could remedy these shortcomings. It appears that the authors could mine some of their own data more deeply.\nA critical question for open data in every area, including agriculture/nutrition, is to identify exactly what kinds of datasets are most valuable and measure the impact of applying that data. In fact, GODAN has recognized the need to help its partners focus on key datasets, and the open data package in beta (reference 7) is designed to do this. The questionnaire used in this study (presented in a supplementary file) had two useful questions at the end about the perceived impact and potential impact of data from different sources. The authors should present findings from those questions, both by supplying the underlying data (which appears to be missing) and their analysis of its meaning.\nIn addition, the authors could make an important contribution to the literature by mining their partners' responses for concrete examples of agriculture and nutrition open data applications. Programs such as the Open Data 500 and the Open Data Impact Map have created databases with hundreds examples of open data projects in all sectors. The authors must have collected a number of valuable examples through their survey, but don't seem to have made that information accessible in an easily usable form. If they could do so, these other programs would be able to integrate their findings and improve the knowledge base for this information.\nFinally, the literature review is not adequate: Almost all the sources cited are GODAN's sources. The authors should provide a richer context by citing and discussing key sources on open data impact, cost analyses, studies of open data for development, and analyses of trends in applying open data for agriculture and nutrition.\nIn sum, this is a useful study that seems to have collected more data than the authors have analyzed and reported on. Additional work to mine the data and present it in context would make this a stronger and more valuable contribution.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-47
https://f1000research.com/articles/7-46/v1	11 Jan 18	{ "type": "Case Report", "title": "Case Report: Non-infectious causes of palmoplantar rashes, what to consider", "authors": [ "Rashmi Advani", "Danit Arad", "Rashmi Advani" ], "abstract": "Background: Palm and sole skin eruptions have a broad differential diagnosis. It is particularly important to recognize common causes as well as their association with certain chemotherapy regimens such as Capecitabine. Case report: A 79-year-old woman presented with a painful rash on her hands and feet for 1 week. She had metastatic colon cancer and was in her third week of treatment with capecitabine. Her diagnosis was a medication side-effect from chemotherapy. Capecitabine was stopped and she had some clinical improvement over the next two days. She was discharged with oncology follow up for resumption of Capecitabine at a lower dose with improvement in her rash 3 weeks later.  Discussion: Skin rashes are a commonly encountered complaint in patients in the inpatient and outpatient setting. It is important to maintain a broad differential diagnosis in those with rashes of the palmoplantar surfaces of the hands and feet. Recognizing skin changes as a possible manifestation of underlying malignancy or a medication side-effect is key in appropriate diagnosis and treatment.", "keywords": [ "Palmoplantar skin rash", "Medication side-effect", "capecitabine" ], "content": "Introduction\n\nPalmoplantar skin eruption is a commonly encountered diagnosis in the inpatient and outpatient setting. Most likely causes include Type IV hypersensitivity reactions (i.e. contact dermatitis), tinea pedis/manuum, psoriasis, and dyshidrotic dermatitis. These rashes may also be associated with underlying malignancies, especially gastrointestinal malignancies or can be associated with a medication side-effect1. Palmoplantar erythrodysesthesia (PPE), also known as hand-foot syndrome is a toxic, cutaneous side effect of well-associated chemotherapeutic agents, especially capecitabine2–4. The pathophysiology of this condition is not well understood and is an active area of investigation. It is important to recognize this side effect early in patients treated with oral capecitabine chemotherapy and to differentiate it from similar presentations in other disease entities. We present a case of a woman on chemotherapy for metastatic colon cancer with a palmoplantar rash.\n\n\nCase report\n\nA 79- year-old Hispanic woman presented with a one-week history of painful rash on her palms and soles. She reported no recent viral illness, travel, previous rashes, joint pains, new lotion, soap or fabric use. She had metastatic colon cancer previously treated with radiation and hemicolectomy three years prior. She currently completed her second week of Capecitabine therapy (1,250mg/m2 twice a day). She had no other contributory medical history or family history and was on no other medications. She describes never having a similar rash in the past.\n\nOn physical exam, she was afebrile, normotensive and appeared chronically ill. Her palms and soles were tender to touch, erythematous, and diffusely edematous with desquamation over the fingertips and toes (Figure 1 and Figure 2). Biochemical testing including complete metabolic panel and complete blood count were normal. Given her recently administered chemotherapy, it was suspected that the patients’ palmoplantar rash was a result of a medication side-effect from Capecitabine. Other less likely diagnoses were contact dermatitis, tinea pedia/mannum, or dyshidrotic dermatitis.\n\nAfter Capecitabine was stopped, she had mild clinical improvement over the next two days. She was discharged with resumption of Capecitabine at a lower dose (565 mg/m2 twice daily) and had complete clinical resolution of her rash 3 week later.\n\n\nDiscussion\n\nPalmoplantar skin eruption carries a varied differential diagnosis. Common causes include contact dermatitis, tinea pedis/manuum, psoriasis, dyshidrotic dermatitis and palmoplantar pustulosis. Other palmoplantar rashes such as palmoplantar keratoderma (PPK), Acanthosis Nigricans (AN), Tripe palm, and Acquired Ichthyosis are also associated with underlying malignancies5,6. PPK presents with a yellow, wax-like hyperkeratosis of the palms and soles. AN, seen in patients with insulin resistance, presents as palmoplantar plaques which can be a sign of internal gastric cancer6. Tripe palm, also associated with gastric and lung malignancies, presents with wrinkled velvety hyperkeratosis of the palmoplantar surfaces7. Lastly, acquired ichthyosis is a symmetric scaling of the skin, associated with Hodgkins lymphoma8.\n\nSince our patient had a temporal relationship between initiation of a new medication and her presentation, it was likely related, if not the cause of her palmoplantar rash. Chemotherapy, such as Capecitabine, is an important cause of palmoplantar skin eruption known as palmoplantar erythrodysesthesia (PPE). It is characterized by pain, swelling and desquamation, which can progress to ulceration and blistering (Figure 1 and Figure 2). In total, 7% of patients treated with Capecitabine may experience PPE. Other commonly encountered chemotherapy regimens may also cause PPE, such as Cytarabine, Fluorouracil, and Doxorubicin. Treatments include either withdrawal of the chemotherapy or dose reduction, and supportive measures. In our patient’s case, we were limited by not being able to completely stop chemotherapy given her limited therapeutic options; however resuming treatment at a lower dose helped to resolve her symptoms as well as provide a longer life-expectancy.\n\nCommon diagnoses aside, medication side-effect and malignancy should be considered in the differential diagnosis of palmoplantar skin eruption to guide appropriate therapy.\n\n\nConsent\n\nWritten informed consent was obtained from the patient for the publication of the patient’s clinical details and accompanying images.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nReferences\n\nThiers BH, Sahn RE, Callen JP: Cutaneous manifestations of internal malignancy. CA Cancer J Clin. 2009; 59(2): 73–98. PubMed Abstract \| Publisher Full Text\n\nNagore E, Insa A, Sanmartín O: Antineoplastic therapy-induced palmar plantar erythrodysesthesia (‘hand-foot’) syndrome. Incidence, recognition and management. Am J Clin Dermatol. 2000; 1(4): 225–234. PubMed Abstract \| Publisher Full Text\n\nNikolaou V, Syrigos K, Saif MW: Incidence and implications of chemotherapy related hand-foot syndrome. Expert Opin Drug Saf. 2016; 15(12): 1625–1633. PubMed Abstract \| Publisher Full Text\n\nKang YK, Lee SS, Yoon DH, et al.: Pyridoxine is not effective to prevent hand-foot syndrome associated with capecitabine therapy: results of a randomized, double-blind, placebo-controlled study. J Clin Oncol. 2010; 28(24): 3824–3829. PubMed Abstract \| Publisher Full Text\n\nEhst BD, Minzer-Conzetti K, Swerdlin A, et al.: Cutaneous manifestations of internal malignancy. Curr Probl Surg. 2010; 47(5): 384–445. PubMed Abstract \| Publisher Full Text\n\nKrawczyk M, Mykała-Cieśla J, Kołodziej-Jaskuła A: Acanthosis nigricans as a paraneoplastic syndrome. Case reports and review of literature. Pol Arch Med Wewn. 2009; 119(3): 180–183. PubMed Abstract\n\nCohen PR, Grossman ME, Almeida L, et al.: Tripe palms and malignancy. J Clin Oncol. 1989; 7(5): 669–678. PubMed Abstract \| Publisher Full Text\n\nRiesco Martínez MC, Muñoz Martín AJ, Zamberk Majlis P, et al.: Acquired ichthyosis as a paraneoplastic syndrome in Hodgkin’s disease. Clin Transl Oncol. 2009; 11(8): 552–553. PubMed Abstract \| Publisher Full Text" }	[ { "id": "30835", "date": "21 Feb 2018", "name": "Sidharth Sonthalia", "expertise": [ "Reviewer Expertise Systemic dermatology", "dermoscopy", "pigmentary disorders" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title could have been better - \"Non-infectious causes of palmoplantar rashes, what to consider\" would have been more apt in a case where there was diagnostic confusion based on history and/or examination. In this case, the history of malignancy and the ongoing chemotherapy was forthcoming. Emphasizing upon the fact that one MUST investigate for concurrent malignancy and chemotherapy, to consider non-infectious cause of the hand and foot syndrome (HFS) in a patient who is presenting with the classical clinical features of HFS and history of capecitabine, the most common chemotherapeutic agent responsible for this condition.\n\nAlthough, despite a strong evidence (history of malignancy and capecitabine) of the etiology of palmo-plantar erythrodysesthesia or HFS, the authors mention that they were considering other less likely diagnoses such as contact dermatitis, dyshidrotic eczema and tinea pedis/manuum. But no confirmatory investigations were done to rule them out. A 10% KOH smear and a punch biopsy should have been taken if these conditions were still being suspected.\n\nIdeally, the authors should have mentioned about the state of the nails of the patient also, which often become dystrophic alongwith HFS.\n\nIt would have been better if the authors had also graded the patient's HFS; in view of the three well-established grading systems in place (WHO, NCI, and one system proposed by Nikolaou et al.1 especially for darker skin types) that have also been documented to guide regarding the therapeutic approach. Similarly, it would have been better if the patient's quality of life (QoL) affected by HFS was quantified using the simple HFS-14 scale. Nikolaou et al have demonstrated that HFS-14 scale may identify differences in QoL impairment between patients with HFS of same clinical grade and may serve as a valuable tool for anticancer treatment management and assessment of clinical efficacy of treatments used for this condition.\n\nThe authors should have at least made a mention of an important differential of HFS, i.e. HFS reaction (HFSR) that predominantly involves the pressure-prone areas of the soles and typically arises as a reaction to multiple kinase inhibitors (MKI).\n\nSince the HFS was not quantified, nor the patient's morbidity due to pain (limitation of instrumental vs self-care activities) has been mentioned, based upon the relatively scarce data on physical examination, the patient seems to qualify for NCI grade 2 HFS. The approach to management in grade 2 has been recommended to stop the chemotherapy for a short duration till the condition improves to grade 1 or 0 (which was done in the current case) followed by resumption at lower doses. However, there are many practical measures and simple drugs/topical applications which if followed could have expedited the improvement such as avoidance of mechanical stress, use of cold compresses, generous moisturization with a urea-containing cream, and potent topical corticosteroids for a short duration. In fact prophylactic oral pyridoxine and celecoxib (COX-2 inhibitor) have also been suggested for a patient with NCI grade 2 HFS.\n\nIt is true that in this particular patient, as per the authors' description, uneventful remission occurred within 2-3 weeks of 2-day cessation followed by resumption at low doses of capecitabine, the report would have given out a more wholesome message to the readers if the treatment modalities other than the stoppage of the chemotherapeutic agent were at least mentioned.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [] }, { "id": "30831", "date": "27 Feb 2018", "name": "Mohamed Badawy Abdel-Naser", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report is well written.\n\nThe title is not addressing the case. Perhaps it can be reformulated to directly refer to the case.\n\nThe statement \"however resuming treatment at a lower dose helped to resolve her symptoms as well as provide a longer life-expectancy\" - how does reduction of the dose provide longer life-expectancy?\n\nThe provided figures, particularly Figure 2, are not clear.\n\nWas any local treatment given to the patient?\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Partly\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "30832", "date": "05 Mar 2018", "name": "Regina Fölster-Holst", "expertise": [ "Reviewer Expertise Pediatric dermatologist", "dermatologist" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe a 79-year old woman presented with palmoplantar rash. This was characterized by painful erythema, edema and desquamation. In this case the history led to the right diagnosis: the woman suffered from metastatic colon cancer and underwent chemotherapy with capecitabine.\nThere are other diagnoses to be considered in patients with these clinical findings. Only some of them are mentioned by the authors. For the readers a table with differential diagnoses and typical clinical and anamnestic criteria would have been helpful to differentiate between these diagnoses.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-46
https://f1000research.com/articles/7-40/v1	10 Jan 18	{ "type": "Research Article", "title": "A SNP resource for studying North American moose", "authors": [ "Theodore S. Kalbfleisch", "Brenda M. Murdoch", "Timothy P. L. Smith", "James D. Murdoch", "Michael P. Heaton", "Stephanie D. McKay", "Brenda M. Murdoch", "Timothy P. L. Smith", "James D. Murdoch" ], "abstract": "Background: Moose (Alces alces) colonized the North American continent from Asia less than 15,000 years ago, and spread across the boreal forest regions of Canada and the northern United States (US). Contemporary populations have low genetic diversity, due either to low number of individuals in the original migration (founder effect), and/or subsequent population bottlenecks in North America. Genetic tests based on informative single nucleotide polymorphism (SNP) markers are helpful in forensic and wildlife conservation activities, but have been difficult to develop for moose, due to the lack of a reference genome assembly and whole genome sequence (WGS) data. Methods: WGS data were generated for four individual moose from the US states of Alaska, Idaho, Wyoming, and Vermont with minimum and average genome coverage depths of 14- and 19-fold, respectively. Cattle and sheep reference genomes were used for aligning sequence reads and identifying moose SNPs. Results: Approximately 11% and 9% of moose WGS reads aligned to cattle and sheep genomes, respectively. The reads clustered at genomic segments, where sequence identity between these species was greater than 95%. In these segments, average mapped read depth was approximately 19-fold. Sets of 46,005 and 36,934 high-confidence SNPs were identified from cattle and sheep comparisons, respectively, with 773 and 552 of those having minor allele frequency of 0.5 and conserved flanking sequences in all three species. Among the four moose, heterozygosity and allele sharing of SNP genotypes were consistent with decreasing levels of moose genetic diversity from west to east. A minimum set of 317 SNPs, informative across all four moose, was selected as a resource for future SNP assay design. Conclusions: All SNPs and associated information are available, without restriction, to support development of SNP-based tests for animal identification, parentage determination, and estimating relatedness in North American moose.", "keywords": [ "Alces", "moose", "single nucleotide polymorphism", "whole genome sequence", "SNP", "parentage", "animal identification", "genetic diversity", "DNA testing", "cattle genomes", "sheep genomes", "wildlife comparative genomics" ], "content": "Introduction\n\nAlces alces is the largest member of the Cervidae family, and ranges throughout the circumpolar boreal forests of Eurasia and North America1,2. The species diverged from the ancestors of domestic cattle and sheep approximately 27 million years ago3. Moose are important ecologically, as a large ungulate with strong ecosystem impacts; economically, due to their value for tourism and hunting; and culturally, as a prominent symbol in many regions4. Consequently, there is active management of moose populations by wildlife agencies throughout their range in North America. However, management is hampered by a lack of genetic tools for monitoring moose, assessing the genetic health of populations, and even detecting illegal harvesting. Moose populations appear to be declining in some regions, including parts of the Upper Midwest of the United States5,6, and effective management is often dependent on data that are logistically challenging and/or costly to collect.\n\nIdentifying individual animals and measuring relatedness among and within populations are important for effective wildlife management and conservation efforts7–9. Identifying individuals can be as simple as observing their unique color patterns, for example in wild dogs (Lycaon pictus)10. However, this is not practical in species such as moose that have few features with obvious variation between individuals. Moreover, coat color patterns provide little information about genetic relatedness. Association of younger and older animals has been used to infer relationships, for example in swift foxes (Vulpes velox) where pups at a den are presumed to be offspring of the attending parents based on the monogamous behaviors they exhibit. However, detailed parentage studies have revealed multiple paternity within swift fox litters11 and other fox species12. Generally, genetic testing provides a more accurate assignment of parentage and supports unique identification of individuals in the vast majority of instances, as well as an estimation of intra- and inter-population genetic variability.\n\nGenetic testing using DNA markers has been applied to human, livestock, and wildlife studies for many years13–16. This form of testing first gained popularity with the development of microsatellite short tandem repeat (STR), and mitochondrial genome markers, concurrent with the development of DNA amplification and sequencing technologies. Approximately 5 to 11 microsatellite markers from cattle, sheep, and caribou (Rangifer tarandus) have been adapted for moose studies17–22. These studies form the basis of our current understanding of North American moose population structure and genetic diversity. DNA technology developments in the past decade have led to the replacement of microsatellite and mitochondrial genome markers with SNP markers because SNPs are more abundant, have greater stability over generations, are more accurately genotyped, and are amenable to automating the genotyping processes23. Moreover, panels of SNPs broaden the use of genotyping for management and conservation efforts, because they can provide not only identification of individuals and parentage, but also estimation of inbreeding and relatedness, and detection of admixture between populations of wildlife. For example, an SNP-based approach has been used for conservation efforts in endangered species such as the Iberian lynx (Lynx pardinus)24 and Tasmanian devil (Sarcophilus harrisii)25, as well as more common but wide-ranging species like the brown bear (Ursus arctos)26. The application of SNP-based approaches, however, requires first the identification of polymorphisms segregating in the populations being studied, and developing assays that support accurate genotyping. Accordingly, a SNP panel spanning the genome of North American moose would be useful for addressing fundamental questions about population genetics in this species.\n\nLow genetic diversity among North American moose populations has been previously reported17,18, making development of SNP panels challenging. The low diversity has been attributed in the prevailing theory, to a relatively recent (ca. 11,000–14,000 years ago) colonization from Asia and subsequent founder effect induced by extended range expansion from an original small group of animals27. However, no definitive evidence has been presented that refutes an alternative hypothesis, that North American moose experienced a severe population bottleneck at some time in the past20, as occurred for North American bison (Bison bison) populations28. Whether a founder effect, bottleneck, or both, the small effective population size simultaneously increases the need for developing genetic tools for management and the challenge of creating SNP marker panels.\n\nDiscovery of SNPs in a species has generally been preceded by development of its reference genome assembly. Using this assembly, whole genome sequence (WGS) reads from individual animals can be aligned, and differences between segregating alleles identified. However, creation of a reference assembly still represents a significant barrier for most research communities interested in wildlife species. Fortunately, an alternative approach that uses the reference genomes of related species has been developed29, and shown to effectively identify high-confidence SNPs likely to be segregating within the target species. Here we report the whole genome sequencing of four moose genomes, each obtained from distant geographic regions of North America, and the use of the cattle and sheep reference genomes to align the sequence data and identify SNPs likely to be segregating among moose populations. A set of criteria was developed to select the potentially most useful set of moose SNPs, and to identify 317 autosomal variants meeting these criteria. The associated sequence information was made freely available, and represents a resource for developing genotyping assays to support moose genetic research.\n\n\nMethods\n\nThis article contains no studies performed with animal subjects, and thus, no additional institutional ethical permits were required. Samples for DNA extraction were donated by private individuals not associated with this research. These were hunters that had legally harvested moose during the firearm hunting season in their state. No additional approvals were needed, since all hunters obtained valid hunting licenses for the harvesting of moose.\n\nSamples of muscle tissue were obtained from four animals likely comprising three putative subspecies of A. alces based on their location in North America: A. gigas, A. shirasi, and A. americana30. These animals were harvested at four distinct geographic locations (Figure 1) and entered as BioSamples in NCBI BioProject Accession PRJNA325061 (Table 1). As is typical, hunters removed the internal organs in the field, the carcasses were chilled, and the meat was subsequently processed for frozen storage. Each of the four owners donated approximately 50 g of frozen tissue from their harvested animal, and that tissue was archived at USMARC for use in this project.\n\nKey:\n\naBased on the observed linear relationship between aligned read depth (y) and total gigabases (Gb) of genomic sequence collected (x) with a quality score ≥ to 20, where y = 0.3x in cattle and sheep genomes38,39.\n\nbNCBI BioProject number 325061\n\ncNCBI BioProject number 324822\n\ndNot determined\n\neNCBI BioProject number 324837\n\nDNA was extracted from muscle with a typical phenol:chloroform method and stored at 4°C in 10 mM TrisCl, 1 mM EDTA (pH 8.0) as previously described31. Approximately 5 μg of moose genomic DNA was fragmented by focused-ultrasonication to generate fragments less than 800 bp long (Covaris, Inc. Woburn, Massachusetts USA). These fragments were used to make an indexed, 500 bp paired-end library according to the manufacturer’s instructions (TruSeq DNA PCR-Free LT Library Preparation Kits A and B, Illumina, Inc., San Diego, California USA). After construction, indexed libraries were pooled with other indexed samples in groups of four to eight, and sequenced with a massively parallel sequencing machine and high-output kits (NextSeq500, two by 150 paired-end reads, Illumina Inc.). After sequencing, the raw reads were filtered to remove adaptor sequences, contaminating dimer sequences, and low-quality reads. Pooled libraries with compatible indexes were repeatedly sequenced until a minimum of 40 Gb of sequence with greater than Q20 quality was collected for each animal. Previous results showed that this level of coverage provided genotype scoring rates and accuracies that exceeded 99%29.\n\nThe DNA sequence alignment process was similar to that previously reported29. Briefly, FASTQ files corresponding to a minimum of 40 Gb of Q20 sequence were aggregated for each animal. The reference assemblies for both UMD3.132 and Oar_v3.1 were downloaded from the NCBI genomes download site and indexed for use with the Burrows Wheeler aligner (BWA) version 0.7.1233. The fastq files corresponding to R1 and R2 runs for the paired end libraries of each respective animal were aligned individually using the BWA aln algorithm and bovine reference assembly UMD3.1. The R1 and R2 datasets were then merged and collated using BWA sampe. The process was repeated for the mapping of the reads to the ovine Oar_v3.1 reference assembly. The resulting sequence alignment map (SAM) files were converted to binary alignment map (BAM) files, and subsequently sorted using Samtools (version 0.1.18)34. PCR duplicates were marked in the BAM files using the Genome Analysis Toolkit (GATK, version 1.5-32-g2761da9)35. Regions in the mapped dataset that would benefit from realignment due to small insertions and deletions were identified using the GATK module RealignerTargetCreator, and realigned using the module IndelRealigner. The BAM file produced at each of these steps was indexed using Samtools. The resulting indexed BAM files were made available via the Intrepid Bioinformatics genome browser http://www.intrepidbio.com/, with groups of animals linked at the USMARC WGS browser (mapped to cattle, mapped to sheep). The raw reads were deposited at NCBI BioProject Accession PRJNA325061. Some SNP variants were identified manually by inspecting the target sequence with Integrative Genomics Viewer (IGV) software version 2.1.2836,37, as described in previously38. In these cases, read depth, allele count, allele position in the read, and quality score were taken into account when the manual genotype determination was made.\n\nThe above mapping efforts produced BAM files for the alignments to both UMD3.1, and Oar_v3.1. The BAM files for all four animals were analyzed simultaneously for variation against both the UMD3.1 and the Oar_v3.1 genomes. The GATK UnifiedGenotyper was used with the genotype mode (-gt_mode) flag set to DISCOVERY, and the likelihood model (-glm) flag was set to BOTH in order to identify both single nucleotide variants, and small insertions and deletions. The maximum number of alternate alleles (--max_alternate_alleles) flag was set to allow only three. Other than those mentioned, default parameters were used. Samtools was used to generate a pileup file containing the measured allele and depth of coverage at each position for all four animals. Variant sites in the four moose were filtered for having a minimal read depth of ten, and a minimum genotype quality score of 30. The SNPs were filtered for having a minor allele frequency (MAF) of 0.5, with both homozygous genotypes present among four animals. Fifty bases of flanking DNA sequence on either side of the targeted moose SNP were analyzed for nucleotide alleles that were homozygous in all four moose yet different from the cattle or sheep reference sequences. These nucleotide sites were flagged as potential moose “species-specific” alleles and the 101 bp of context sequence was edited to create a moose consensus reference sequence. The 101 bp of moose consensus sequence derived from the alignment of one reference genome was then tested for alignment to the other reference genome. Moose SNPs with MAFs of 0.5, and having been derived independently from alignment to both reference genomes, were manually assigned genome-wide bins based on their chromosome and proximity as inferred by alignment with the cattle genome. The goal of assigning markers to bins was to minimize linkage while allowing automated SNP assay design software the opportunity to select the best candidate marker for each distinct genomic region. All of these conservative filters were intended to maximize marker informativity in North American moose populations, and minimize potential technical difficulties with SNP assay designs that rely on oligonucleotide hybridization for genotype detection.\n\n\nResults\n\nAn average of 63.5 Gb total genome sequence was collected for four moose. Based on similar estimates in cattle and sheep, this would correspond to an average read depth of 19-fold coverage if aligned to a moose reference genome of similar quality (Table 1). However, when cattle and sheep reference genomes were used, an average of 11.0 and 8.7% of the moose reads were aligned, respectively. For comparison, the same alignment method was performed with sets of bovine and ovine genomic sequences and resulted in 88.4% and 83.8% reads aligned to their respective genome assemblies (Table 1). For cross-species comparison, 22.2% of the ovine set of genomic sequence reads were aligned to the bovine assembly. Although the moose read depth was low when averaged across the entire genome of cattle or sheep, at conserved genome regions it was consistent with the expected average read depth of 19-fold. Thus, the moose read depth in conserved genomic regions appeared to be sufficient for identifying polymorphic sites and accurately assigning variant alleles.\n\nAlignment of moose reads to the cattle and sheep genomes identified approximately 48.3 million and 39.7 million sites that differed from the reference assemblies, respectively. These included SNPs, insertions and deletions, and sites where moose-associated nucleotide differences occurred. The latter sites were defined as having homozygous genotypes in the four moose, with alleles differing from those in cattle or sheep (Figure 2). After stringent filtering for read depth and alignment quality, there were 1,095,371 and 813,006 moose variants identified with the respective cattle and sheep genome assemblies (Table 2). Approximately 96% of these were homozygous moose-associated nucleotide differences (Supplementary file S1 and Supplementary file S2). The remaining 46,005 and 36,934 variants were moose SNPs identified by the respective cattle and sheep alignments (Supplementary file S3 and Supplementary file S4). The MAF distribution of the moose SNPs was similar for both sets with the large group having a 0.125 MAF (approximately 37%, Table 2). The most informative moose SNPs (i.e., “highly informative”) were defined as those with a 0.5 MAF and both homozygous genotypes present among any of the four moose, and are candidate SNPs that may have arisen to a high MAF prior to the species arrival in North America (Figure 3). There were 1,341 and 1,014 of these moose SNPs identified with the cattle and sheep alignments, respectively (Table 2).\n\nOverlapping computer screen images of moose WGS data aligned to bovine and ovine reference genomes, respectively, showing two moose-associated nucleotides in the GDF9 gene. The bovine UMD3.1 and ovine Oar_v3.1 map positions for the variant sites are chr7:46,065,076 - 46,065,079 and chr5: 41,841,536 - 41,841,536,539, respectively.\n\nKey:\n\naAutosomal chromosome alignment with minimum read depth of ten and minimum genotyping quality score of 30. There were approximately 60,600 and 58,200 additional moose SNPs in the respective UMD3.1 and Oar_v3.1 alignments that were heterozygous in all four moose. However, these were excluded from the SNP counts because this artifact is caused by sequence read misalignment.\n\nbThese sites are difference from the reference and homozygous in all four moose.\n\nc101 bp flanking regions of SNPs with 0.5 MAF that could be unambiguously identified by BLAT alignment to the other reference assembly (Table S1 and Table S2). These regions also contained no SNPs among the four moose.\n\ndSNP that were independently identified by alignment in each reference genome and manually grouped into 216 chromosomal bins for assay design (Table S3).\n\nOverlapping computer screen images of moose WGS data aligned to bovine and ovine reference genomes, respectively, showing a highly informative SNP. Screenshots from IGV software showing one of 317 moose SNPs with a 0.5 MAF, both homozygous genotypes present, and aligned to genomic regions conserved in all three species. The bovine UMD3.1 and ovine Oar_v3.1 map positions are chr1:7,634,617 and chr1: 126,412,162, respectively.\n\nCandidate moose SNPs were further excluded when the flanking sequences in one reference genome were not uniquely identified in the other. This left 773 and 552 highly informative moose SNPs identified in conserved regions of the cattle and sheep genomes, respectively (Supplementary Table S1 and Supplementary Table S2). Of these 1,325 highly informative SNPs, 1,008 were unique between the two sets, while 317 were common to both sets. The latter represents the most informative moose SNPs, with the highest flanking sequence conservation, due to their independent alignment to both reference genomes (Table 2).\n\nThe alignment coordinates of the 1,325 highly informative SNPs were analyzed for genome-wide distribution patterns that may indicate ascertainment biases caused by the variant selection. Overall, the distribution of SNP sites in the sets with 773, 552, and the 317 intersecting markers, appeared to be widespread in the cattle and sheep genomes and generally appropriate for genome-wide estimates (Figure 4). However, some SNP clustering was observed as the set of 317 had a mean and median spacing of 5.3 and 2.1 Mb, respectively (Supplementary Figure S1A). To facilitate SNP genotype assay design, the clustered SNPs were manually grouped into 216 bins with a mean size of approximately 8.1 Mb (median 5.9 Mb, Supplementary Figure S1B). Thus, SNP assay designs could be directed to each bin, with the option to use any SNP from that bin for multiplex assay design (Table S3).\n\nThe distribution of moose SNPs with 0.5 MAF relative to the cattle and sheep chromosomal locations (see Table S1 and Table S2 for marker details). The inset shows the chromosomal map positions with the cattle UMD3.1 reference assembly.\n\nGenotype analysis for the 773 and 552 moose SNPs derived from the cattle and sheep alignments, respectively, showed that each moose had approximately the same proportion of opposing homozygous genotypes (Figure 5A, Supplementary Table S4 and Supplementary Table S5). However, there were significant differences in the ratio of heterozygous genotypes to homozygous genotypes (Figure 5B). The Alaskan moose had the most favorable average heterozygosity ratio (1.26), followed by the moose from Wyoming and Idaho (1.10 and 1.06, respectively), and the Vermont moose (0.68). Note that the numerical value of the ratios calculated from these SNP is likely an underestimate of the within-animal genome-wide heterozygosity, because there may be ascertainment bias resulting from targeting of SNP discovery to genomic regions conserved between three species. The SNP allele sharing between each of the four moose was analyzed with the sets of 773 and 552 markers to obtain a genome-wide measurement of their relatedness. This was possible because the method for selecting each of these SNPs was not dependent on which two of the four moose were heterozygous. The pair of moose from Alaska and Idaho had the highest proportion of shared alleles (0.430 and 0.397), while the Alaska and Vermont pair had the lowest (0.255 and 0.279, Table 3). Together, the genotype results with these sets of 773 and 552 SNPs indicate that there was a west-to-east pattern of decreasing genetic diversity in the four moose used in this study.\n\nThe ratio of heterozygous to homozygous genotypes from four moose was evaluated with sets of 773 and 552 markers SNPs with 0.5 MAF (see Table S1 and Table S2 for marker details). (A) Genotype counts for each of the four animals with the 773 moose SNPs identified in the alignment to cattle (Table S1) and the 552 moose SNPs identified in the alignment to sheep (Table S2). (B) The heterozygosity ratios calculated for each of the four animals from the 773 SNP set (grey circles); and the 552 SNP set (tan circles). The ratio consisted of the number of heterozygous sites divided by the combined number of homozygous sites.\n\nThe combined set of 1,008 highly informative moose SNPs were also evaluated for their relative proximity to genes in the annotated reference assemblies of cattle and sheep. In the sets of 773 and 552 moose SNPs, 256 and 181 were present within genes, respectively (Table S6 and Table S7). Some genes contained more than one polymorphism, and thus, there were 221 and 178 total cattle and sheep genes, respectively, with highly informative SNPs. Of these genes with moose SNPs, 84 were identified in both cattle and sheep alignments. In addition, there were a number of informative SNPs in noteworthy genes that did not pass the read depth and quality score filters. For example, the prion gene (PRNP) affects susceptibility to spongiform encephalopathies such as chronic wasting disease in cervids. By manually viewing the PRNP coding sequence with IGV software, a coding SNP with 0.5 MAF and both homozygotes present was identified (M217I, Table 4). Thus, the publicly searchable and viewable moose WGS presented here represents a novel genomics resource that may facilitate candidate gene-based research in this species.\n\nKey:\n\naBased on samples from four individuals sourced from Alaska (AK), Wyoming (WY), Vermont (VT), and Idaho (ID), USA.\n\nbHighly-informative moose parentage SNPs with 0.5 MAF and both homozygous genotypes present among the four moose.\n\ncHomozygotes are denoted with the one-letter nucleotide code. Heterozygotes are denoted with IUPAC/IUBMB ambiguity codes: R = a/g, Y = c/t, M = a/c, K = g/t, S = c/g, W = a/t.40.\n\ndThe PRNP codon number 217 refers to the number system in cattle. In moose, this codon is at position 209.\n\neThe IGF1 codon is in exon 2 and the numbering for codon 27 is the same in cattle as in moose.\n\n\nDiscussion\n\nWe sequenced four moose from regions that span the United States, to approximately 19-fold genome coverage, and aligned them to the cattle and sheep reference genomes. Approximately 10% of moose sequences were aligned and used to identify more than 40 k moose SNPs in this cross-species approach. The relatively low alignment rate may be a reflection of the 27 million year average molecular divergence time between moose and non-cervid members of the Pecora infraorder3. In spite of the alignment rate, 1,008 highly informative moose SNPs were identified for future use in developing DNA-based genetic tests to support forensic and wildlife conservation activities. These 1,008 moose SNPs were derived from the intersection of two overlapping sets aligned to cattle (773 SNPs) and sheep (552 SNPs) reference genome assemblies. The 1,008 moose SNPs were refined to a minimal subset of 317 moose SNPs found in the most highly conserved genome regions. All of these markers are publicly available and ready for validation on a variety of SNP genotyping technology platforms. An important first step in evaluating these SNPs will be characterizing their MAFs in wild populations of North American moose. The online whole genome moose sequences, together with reference genotypes (Supplementary Table S4 and Supplementary Table S5) and DNA from these four moose, provide the opportunity for immediate design, testing, and validation of these candidate parentage SNPs.\n\nGenotype information from the 1,008 moose SNPs was useful for measuring genome-wide differences in DNA sequence diversity among the four individuals. Measurements of heterozygosity and allele sharing showed that the Alaskan moose was the most diverse, the Vermont moose was the least, with the moose from Idaho and Wyoming being intermediate. This is consistent with a species that crossed the Bering Land Bridge into Alaska and radiated outward from west to east across North America. SNPs have been previously used to estimate genome diversity in other species with low genetic diversity like the European bison (B. bonasus)41 and the Tasmanian devil (S. harrisii)25. A caveat with our results is the overall heterozygosity of each moose may be underestimated due to ascertainment bias for highly informative SNPs in highly conserved genomic regions. In other words, variation in conserved moose genome regions may occur at a lower rate than that in non-conserved regions. In spite of this potential ascertainment bias, the results suggest that combinations of these markers may be useful in detecting population structure.\n\nAn important unanswered question is: how informative will these SNPs be in moose populations? Population-wide data to address this question will require development and application of genotyping assays, and assembly of pertinent samples for testing, which was beyond the scope and resources of the present report. The data presented here, which identify polymorphisms with alternate homozygous genotypes in a limited sample of only four individuals, suggest that the SNP selected represent variation that existed prior to arrival of moose in North America.\n\n\nConclusions\n\nThese moose SNPs and associated sequence information are available for use without restriction, and provide a basis for developing commercial SNP-based “parentage” SNP DNA tests for validation in North American moose populations.\n\n\nData availability\n\nFASTQ files for the four moose combined are available in NCBI SRA, with contiguous accession numbers SRX3218250 - SRX3218281.\n\nThe SRA accession numbers for each individual are:\n\nSRX3218264 - SRX3218271, Alaska moose HM2013;\n\nSRX3218254 - SRX3218259 and SRX3218262 - SRX3218263, Wyoming moose JC2001; SRX3218250 - SRX3218253 and SRX3218272 - SRX3218275, Vermont moose R199; and SRX3218260 - SRX3218261 and SRX3218276 - SRX3218281, Idaho moose Clearwater06.\n\nThe data are part of NCBI BioProject Accession PRJNA325061.\n\nIn addition, access to the aligned sequences is available via the USDA: http://www.ars.usda.gov/Research/docs.htm?docid=25590 (moose aligned to cattle), and http://www.ars.usda.gov/Research/docs.htm?docid=25712 (moose aligned to sheep).\n\nDownload access to the BAM files is available at the Intrepid Bioinformatics sites: http://server1.intrepidbio.com/FeatureBrowser/customlist/record?listid=7919250313 (moose aligned to cattle), and http://server1.intrepidbio.com/FeatureBrowser/customlist/record?listid=7919250315 (moose aligned to sheep).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The USDA is an equal opportunity provider and employer.\n\n\nGrant information\n\nFunding for this research was provided by the USDA, ARS appropriated projects 5438-32000-033-00D and 5438-31320-012-00D, the USDA National Institute of Food and Agriculture, McIntire-Stennis project 1002300, the University of Vermont College of Agriculture and Life Sciences, the College of Agricultural and Life Sciences at the University of Idaho, with the resources of the University of Louisville’s research computing group and the Cardinal Research Cluster.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank J. Carnahan of USMARC for her outstanding technical assistance; H. Moertl (Alaska), B. Carnahan (Wyoming), C. Alexander of the Vermont Fish and Wildlife Department, and Y. Doe and D. Davenport (Idaho) for providing, or assisting with acquisition of moose samples. This work was funded, in part, by the University of Vermont College of Agriculture and Life Sciences, the College of Agricultural and Life Sciences at the University of Idaho, with the resources of the University of Louisville’s research computing group and the Cardinal Research Cluster, and we thank H. Simrall of U. Louisville for his assistance.\n\n\nSupplementary materials\n\nTable S1. List of 773 moose parentage SNPs aligned to bovine reference assembly UMD3.1.\n\nClick here to access the data.\n\nTable S2. List of 552 moose parentage SNPs aligned to ovine reference assembly Oar_v3.1.\n\nClick here to access the data.\n\nTable S3. List of 317 candidate moose parentage SNPs grouped into 216 bins for use in multiplex assay design.\n\nClick here to access the data.\n\nTable S4. Genotypes for 773 moose SNPs aligned to the bovine reference assembly UMD3.1.\n\nClick here to access the data.\n\nTable S5. Genotypes for 552 moose SNPs aligned to the ovine reference assembly Oar_v3.1.\n\nClick here to access the data.\n\nTable S6. List of 256 moose parentage SNPs occurring within genes annotated in bovine reference assembly UMD3.1.\n\nClick here to access the data.\n\nTable S7. List of 181 moose parentage SNPs occurring within genes annotated in ovine reference assembly Oar_v3.1.\n\nClick here to access the data.\n\nFigure S1. Distributions for 317 moose parentage SNPs and their 216 marker groups.\n\nClick here to access the data.\n\nFile S1. VCF file with 1,095,371 moose-specific variants aligned to the cattle UMD3.1 reference assembly.\n\nClick here to access the data.\n\nFile S2. VCF file with 813,006 moose-specific variants aligned to the sheep Oar_v3.1 reference assembly.\n\nClick here to access the data.\n\nFile S3. VCF file with 46,005 moose SNPs aligned to the cattle UMD3.1 reference assembly.\n\nClick here to access the data.\n\nFile S4. VCF file with 36,934 moose SNPs aligned to the sheep Oar_v3.1 reference assembly.\n\nClick here to access the data.\n\n\nReferences\n\nFranzmann AW: Alces alces. Mammalian Species. 1981; 154: 1–7. Publisher Full Text\n\nKarns PD: Population distribution, density and trends. In: Franzmann AW, Schwartz CC, editors. Ecology and management of the North American moose. Washington, D.C., USA: Smithsonian Institution Press, 1998; 125–39.\n\nKumar S, Stecher G, Suleski M, et al.: TimeTree: A Resource for Timelines, Timetrees, and Divergence Times. Mol Biol Evol. 2017; 34(7): 1812–9. PubMed Abstract \| Publisher Full Text\n\nTimmermann HR, Rogers AR: Moose: competing and complementary values. Alces. 2005; 41: 85–120. Reference Source\n\nLenarz MS, Fieberg J, Schrage MW, et al.: Living on the edge: viability of moose in northeastern Minnesota. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeaton MP, Smith TP, Carnahan JK, et al.: Using diverse U.S. beef cattle genomes to identify missense mutations in EPAS1, a gene associated with high-altitude pulmonary hypertension [version 1; referees: 2 approved]. F1000Res. 2016; 5: 2003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHeaton MP, Smith TP, Freking BA, et al.: Using sheep genomes from diverse U.S. breeds to identify missense variants in genes affecting fecundity [version 1; referees: 2 approved]. F1000Res. 2017; 6: 1303. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNC-IUB: Nomenclature for incompletely specified bases in nucleic acid sequences. Recommendations 1984. Nomenclature Committee of the International Union of Biochemistry (NC-IUB). Proc Nat Acad Sci U S A. 1986; 83(1): 4–8. PubMed Abstract \| Free Full Text\n\nTokarska M, Marshall T, Kowalczyk R, et al.: Effectiveness of microsatellite and SNP markers for parentage and identity analysis in species with low genetic diversity: the case of European bison. Heredity (Edinb). 2009; 103(4): 326–32. PubMed Abstract \| Publisher Full Text" }	[ { "id": "30007", "date": "30 Jan 2018", "name": "Joshua M. Miller", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this work Kalbfleisch et al. develop a novel set of SNP loci for moose. To do so the authors generated genomic sequence data from 4 moose across their range in the United States, aligned the reads to both cow and sheep reference genomes, and then applied a series of filters to select a subset of loci in highly conserved regions. The resulting set of loci showed a gradient of diversity decreasing from west to east, consistent with hypothesized colonization. The authors state that these loci will serve as a resource for future management and conservation applications. I think that this study has laudable goals and adds a valuable resource for moose conservation. However, there are some issues the analyses and presentation that need to be clarified.\n\nOverall comments\nThe alignment and filtering procedures seem overly restrictive. Authors note that only ~10% of their reads aligned to the cow and sheep reference genomes (not surprising given the levels of divergence among the species) meaning that 90% of the data was essentially thrown away. Why not start with a de novo assembly of the moose sequence?\nAlong these lines there are several recent papers that detail a hybrid procedure for genome construction that begins with de novo assembly and then apply cross-species alignment for scaffolding, e.g. https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-017-1911-6\n\nIf part of the goals is to eventually use these loci for “population genetic applications” (e.g. looking for population structure and assigning individuals to those populations) it strikes me that selecting loci that are in such heavily conserved regions may result in biased estimates. Can the authors comment on this? Doesn’t this procedure result in a lot of “moose specific” variants being missed? I can understand that it is not the objective of this paper to create a draft genome sequence for the moose (though the authors note in several places that this would be useful, and the data generated here seem appropriate to attempt this), but then more justification as to why this was not attempted needs to be given. Did the authors check and make sure that the “highly conserved genomic regions” are not in repetitive elements? A blast search of these regions should do the trick.\n\nSpecific comments\nIn the sentence in the introduction starting with “DNA technology developments…” saying that SNPs have “replaced” microsatellites and mitochondrial DNA is a gross overstatement. There are many studies still using these markers and there are many applications where these markers may be preferable. Would be better to say something along the lines that SNPs have gained in use. As currently written, the sentence in the introduction starting with “Moreover, panels pf SNPs broaden…” is incorrect. Microsatellites and mitochondrial DNA also provide means of measuring inbreeding and relatedness among individuals. This should be rephrased to state that SNPs may be better at these estimates, likely due to the factors listed in the previous sentence (abundance, accuracy, etc.) I would couch the statement that previous estimates of low genetic diversity in moose will make it hard to discover SNPs. This may be true, but it has not been tested and estimates of diversity from 5-11 microsatellites likely will not reflect genome-wide patterns. Remove “in the prevailing theory,” I find it striking that in the final paragraph of the introduction there is no mention of the plethora of genotype-by-sequencing approaches that are used for SNP discovery and population genetic analyses but do not require a reference genome, e.g Peterson et al. (2012)1. This should be addressed. When you state the sub-species names in the Methods refer readers to Table 1 so that they know which occurs where relative to your samples. Which program or programs were used to filter the raw reads? State what “UMD3.1” and “Oar_v3.1” are when you first introduce them in the methods Is binning relative to the cow genome actually informative for moose? Are the karyotypes comparable? How many chromosomes does the moose have? Along this same line, I think having the variants locations for both UMD3.1 and Oar_v3.1 plotted on top of each other in Figure 4 is misleading. It suggests that the locations are syntenic, which is not true. For instance, the plot implies that no variants were discovered on chromosomes 27, 28, and 29 in the sheep genome, when really this is impossible as sheep only have 27 autosomes. I would suggest re-doing this figure with separate panels for each figure, though see the caveat above. In Figure 5A why are there two bars for each of the “Hom” counts but only one for the “Het”?\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "30045", "date": "30 Jan 2018", "name": "Paul Stothard", "expertise": [ "Reviewer Expertise Genetics", "genomics", "bioinformatics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis well-written manuscript describes the discovery and characterization of single nucleotide polymorphisms (SNPs) in moose. These SNPs will be valuable for future research and conservation efforts, as they can be used for animal identification, assigning parentage, and estimating intra- and inter-population genetic variability. The SNP discovery and genotyping are done using well-established and clearly described approaches, and care has been taken to avoid false positive SNPs (requiring that both homozygous genotypes be observed for example). The raw sequencing data is available through the SRA database, while the aligned reads (BAM) files are available via the USDA and Intrepid Bioinformatics sites. The final filtered variants are provided with flanking sequence in the supplementary materials. Larger, less-filtered collections of variants are provided in the form of VCF files, again in the supplementary materials. The clear, detailed manuscript and the raw data and progressively filtered results will make is easy for others to reproduce or make use of the results of this work.\nMinor comments\n1. In the last paragraph of the introduction the authors note the challenge of creating a whole genome assembly for use in SNP discovery, and that the use of an existing reference genome from a related species can be an effective alternative. In the discussion section the authors mention that the cross-species mapping approach, however, has the drawback of targeting conserved regions of the genome. I am curious as to why the authors chose not to employ a technique like RADseq for SNP discovery, as it does not depend on the availability of a reference genome and would allow them to better assess minor allele frequency, through the inclusion of more individuals. Also, RADseq would be equally effective at targeting conserved and non-conserved regions. The reasons for not using RADseq (or its potential value in future studies) could be addressed in the introduction.\n2. The figure legends for Figure 2 and Figure 3 could be expanded slightly to explain to readers not familiar with IGV which elements represent reads, coverage, reference sequence. Also, in Figure 3 it isn't clear to me why the sequence of one read is shown (moose 4, second read from top, aligned to the sheep reference).\n3. In the Methods section \"fastq\" is written as \"FASTQ\" and \"fastq\".\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "29739", "date": "06 Feb 2018", "name": "Kris J. Hundertmark", "expertise": [ "Reviewer Expertise Moose evolution", "population and spatial genetics of moose and other large mammals" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis study presents SNP discovery and variation of moose in North America, which are very valuable data. First, moose are an understudied species and their genetics can tell us a lot about the phylogeography of species after the LGM. As the authors state, moose in North America have generally low neutral nuclear diversity, and are less diverse than their European counterparts (information from Siberia, if it exists, has not been published in English). They are also less diverse than many northern species and many species of cervids, such as caribou, white-tailed deer, and elk. Hypotheses have been proposed for these observations and have been tested with mtDNA, but only recently have nuclear markers been used to study phylogeography in European moose. No such tests have been conducted in North America. This study provides new tools to spur moose research, although I agree with Reviewer 2 that microsatellites and mtDNA still have their uses.\n\nThe inclusion of 4 individuals in this study is good, as is the broad geographic range of the samples across North America. The methods are appropriate, although as the first reviewer points out, RADSeq could have been used in SNP discovery and has some advantages over the methods used, but I believe that this report on moose is secondary to the authors’ overall goals and we are fortunate that Kalbfleish et al. decided to pursue this and share their findings. The comparison to reference genomes, although from bovids not very closely related to moose, yielded some advantages, including identifying SNPs in important functional genes, such as the PRNP locus. Nonetheless, a white-tailed deer genome was made public in summer 2017 and although these authors may not be willing to start over by comparison of their data to a very similar genome (same subfamily and same number of chromosomes) the prospect exists and should be pursued. I can see the point of Reviewer 2 concerning Fig. 4 and assumed synteny but I believe I got the intended message from the figure as is. But I think mentioning somewhere that North American moose have 34 pairs of autosomes when discussing the success of mapping SNPs to the reference genomes would be appropriate.\n\nI find the last sentence interesting in that the authors believe the SNP variants they found existed prior to moose entering North America. Considering that event likely happened within the last 15,000 years that is a very reasonable statement, and may cause some to think that SNPs may contain little information about geographic variation in moose and all that goes with it. Given the morphological and behavioral differences between, say, Alaskan moose and those in the eastern continent, however, it is obvious that moose have evolved rapidly in that time, despite limited genetic diversity due to Pleistocene bottlenecks and founder effects. How that translates to current SNP diversity and what the latter may be able to tell us about moose are exciting questions to be asked, for which this manuscript sets the stage.\n\nMinor comments:\n\n2nd paragraph of Introduction: should be “among individuals” not “between individuals.”\n\nThird paragraph of Introduction: should be “a SNP-based approach” not “an SNP-based approach.”\n\nLast paragraph of Introduction, last sentence: should it be “has been made freely available” rather than “was made freely available?”\n\nLast paragraph of WGS Production …, last words of 2nd-to-last sentence: Should be “previously.” not “in previously.”\n\nDiscussion, last sentence: should this be “SNPs” instead of SNP?”\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-40
https://f1000research.com/articles/7-39/v1	10 Jan 18	{ "type": "Research Note", "title": "Improving communication for interdisciplinary teams working on storage of digital information in DNA", "authors": [ "Emily E. Hesketh", "Jossy Sayir", "Nick Goldman", "Emily E. Hesketh", "Jossy Sayir" ], "abstract": "Close collaboration between specialists from diverse backgrounds and working in different scientific domains is an effective strategy to overcome challenges in areas that interface between biology, chemistry, physics and engineering. Communication in such collaborations can itself be challenging. Even when projects are successfully concluded, resulting publications — necessarily multi-authored — have the potential to be disjointed. Few, both in the field and outside, may be able to fully understand the work as a whole. This needs to be addressed to facilitate efficient working, peer review, accessibility and impact to larger audiences. We are an interdisciplinary team working in a nascent scientific area, the repurposing of DNA as a storage medium for digital information. In this note, we highlight some of the difficulties that arise from such collaborations and outline our efforts to improve communication through a glossary and a controlled vocabulary and accessibility via short plain-language summaries. We hope to stimulate early discussion within this emerging field of how our community might improve the description and presentation of our work to facilitate clear communication within and between research groups and increase accessibility to those not familiar with our respective fields — be it molecular biology, computer science, information theory or others that might become relevant in future. To enable an open and inclusive discussion we have created a glossary and controlled vocabulary as a cloud-based shared document and we invite other scientists to critique our suggestions and contribute their own ideas.", "keywords": [ "DNA-storage", "digital information storage in DNA", "synthetic biology", "glossary", "communication", "controlled vocabulary", "short plain-language summaries", "interdisciplinary collaboration" ], "content": "Introduction\n\nAs we tackle increasingly complex issues throughout science, a breadth of knowledge is often necessary to devise novel solutions — something frequently achieved through interdisciplinary collaborations. The inherent diversity within interdisciplinary teams stimulates knowledge exchange, creativity or even a change in perspective; however, it can be very challenging. We work within an emerging field in synthetic biology, repurposing DNA as a storage medium for digital information. Advancing from early proof-of-principle studies in the high-throughput era1,2 (see references therein for historical perspective) towards a more reliable, refined and functional large-scale DNA storage system3,4 raises unique challenges that can only be resolved through a broad collaborative effort between biochemical and DNA sequencing specialists, computer and molecular scientists, information theorists and others. This body of research has gained considerable interest both within the research community and with the public, and this has further emphasised the need to address our communication and the presentation of our work.\n\nIntersection between these fields is clearly beneficial. Information theory has already underpinned many advances in life sciences, from adapting Levenshtein coding to create error-correcting molecular barcodes used in multiplexed DNA sequencing5 to Burrows-Wheeler transformation of reference genomes implemented in several short read aligners6–8. A molecular biologist may see the process of storing information in DNA as a very physical process, progressing from DNA synthesis (writing) to amplification (copying) to sequencing (reading). To an information theorist, this is a noisy channel: a series of transformations through which information is transmitted and the outputs observed. Differences in the way experts in these different fields describe their data and results can hinder collaboration and restrict impact. As a result, publications have the potential to be an ineffective hybrid of accepted nomenclature and data presentation within the intersecting fields with few readers, both in the team and outside, able to fully understand the publication as a whole.\n\nUnsurprisingly, common nomenclature between the intersecting disciplines has disparate meanings. Use of the word ‘qubit’ can lead you to believe that some DNA needs quantifying9 or you may be discussing quantum information or quantum field theory10. This complicates communication; misunderstandings have the potential to pass unnoticed, only becoming apparent downstream. Examples of such misunderstandings are the use of the words errors, erasures, and substitutions when retrieving data through DNA sequencing. To an information theorist, an ‘error’ refers to a falsely read symbol, for example when an A in the DNA sequence is falsely read as a C, distinct from an insertion or deletion. An ‘erasure’ would be a read that was possibly so uncertain that it is neither called as an A, C, G or T, but distinct from a ‘deletion’ in that the read is not simply missed but we are made aware that there is a missing symbol at this position in the DNA string. An ‘insertion’ is a symbol read, when no symbol should exist. To a molecular biologist and DNA sequencing expert, all of these would be described as read ‘errors’. To them, errors in the information theoretic sense would be called substitutions.\n\n\nA glossary and controlled vocabulary for DNA-storage\n\nDNA-storage has become a popular research field, with a number of interdisciplinary teams forming and collaborating in an attempt to make viable information storage systems that capitalise on DNA’s numerous advantages11. To alleviate confusion and improve daily communication within and between these groups we propose, and have begun to implement, two measures: a glossary and a controlled vocabulary.\n\nWe have created a glossary defining basic terms in molecular biology, information theory and computer science etc. that are relevant to DNA-storage, for those unfamiliar with one or more of these disciplines. This proved to be a useful aid in early discussions within our team and helped to identify areas of nomenclature ambiguity which if not addressed may have complicated communication downstream. We have already experienced the advantages of sharing this within our team and with collaborators to facilitate exchange of ideas with them.\n\nOur glossary is held on a cloud storage system, and can be found at https://goo.gl/x6B73Q or https://rebrand.ly/dna-storage-glossary. To allow an open and inclusive discussion of how we might improve communication within this emerging community, we encourage others to critique and contribute to the glossary. The document permits “Suggestions” (proposed edits) and “Comments” to be added, and we will review these regularly and update the document as a resource for our research community.\n\nLeading on from this, we are developing an evolving controlled vocabulary allowing team members to communicate precisely. This has been particularly beneficial during technical discussions — for instance, to us data packet refers to part of a DNA sequence that decodes to digital information, and excludes parts that are designed to facilitate DNA sequencing or indexing.\n\nUse of a controlled vocabulary is something that the community may wish to agree upon. For example, one question we pose is — what should we name these DNA sequences that encode digital information? Following the practice of genome scientists, we initially called collections of such DNA sequences libraries. However, working with such samples caused confusion with our colleagues in a molecular biology laboratory: in a Next Generation Sequencing context, the term library is commonly used to describe DNA fragments that have been prepared for DNA sequencing. We now propose to refer to DNA sequences that store digital information as inDNA (for ‘information-carrying DNA’). To refer to inDNA prepared for DNA sequencing, we can now unambiguously talk about a library of inDNA.\n\nWe would like to invite others to contribute to the development of a controlled vocabulary so that we might be able to communicate more precisely. We have included a few entries within our glossary document.\n\n\nImproving review, accessibility and impact of interdisciplinary publications\n\nWe now pose another question — how might we improve data description and presentation to increase accessibility and facilitate peer review and reproducibility? Peer review is crucial within the scientific community, but this quality improvement process may not be fully realised in interdisciplinary publications. We have experienced difficulties with peer review of publications related to DNA-storage applications, as authors of work under review, as reviewers ourselves, in our assessment of others’ reviews, and in dealings with journal editors. Often the expertise is not available, or reviewers may only evaluate limited aspects of the paper. The body of work may not be effectively reviewed as a whole, leaving authors without vital feedback and potentially leading to publication of flawed work.\n\nThe concept of standardising presentation of data and methods is not a novel idea in the life sciences, with ‘minimum information’ standards ensuring that publications contain the information necessary to interpret the experimental data. These are typically technique- or study-specific, e.g. MIAME (microarray experiments)12, MIQE (quantitative polymerase chain reaction)13 and MIFlowCyt (flow cytometry)14. Such an approach may not be appropriate to publications relating to DNA-storage applications for some time, as these typically encompass a number of disciplines, each with its own established data description standards and many of which use rapidly changing technologies. It is not appropriate or practical to standardise such a diverse range of technologies and disciplines. Rather we should respect the accepted discipline norms, blending these together to permit DNA-storage standards to evolve.\n\nEven publications that sit predominantly within a single discipline may be of interest to those unfamiliar with that discipline and benefit from the inclusion of a whole-paper plain-language summary. As standard with plain-language summaries this should simply report the basic rational, methodology and main findings. Box 1 is a whole-publication plain-language summary of 2 that we have written as an example.\n\nWith the amount of digital information that needs to be stored growing exponentially there is a need to develop new ways of storing information. High information capacity, longevity and constant improvements in technologies that allow writing, copying and reading make DNA an attractive medium for storing digital information. Here we present a scalable reliable method for storing digital information in DNA.\n\nThe original bytes of several computer files in various formats were encoded into DNA as follows. A Huffman code was used to compress each byte, depending upon occurrence frequency, into a block of 5–6 trits, which are the characters 0, 1 or 2 (just as bits are 0 or 1). A reference table of these blocks and corresponding nucleotide sequences was created, with each block having four possible nucleotide combination representations. Nucleotide combinations were selected depending also upon the previous block, in a manner that prevented the occurrence of any repeating nucleotides (e.g. AA), as these are known to cause downstream copying and reading problems. Following encoding the digital information was represented as 153,335 DNA sequences of length 117 nucleotides, each containing an index and a simple error checkpoint in addition to encoding part of the original digital information. These DNA sequences were printed as a pool of DNA, containing ~1.2 × 107 copies of each sequence, which was copied via PCR and prepared for reading via DNA sequencing before being decoded (encoding strategy reversed).\n\nData totalling 739 kilobytes was successfully encoded into DNA, printed, copied, read and decoded with 100% accuracy. A storage density of ~2.2PB g−1 DNA was achieved.\n\nIt may also be useful to provide a plain-language summary of a specific technical aspect of a publication. For example, a molecular scientist may not understand the details of a complex mathematical algorithm (and nor should the description be altered specifically to allow them to), but an appreciation of how the output impacts aspects of the project relevant to them may be sufficient. We illustrate this using a paragraph from 4 (from p.5, Methods — Address Design and Encoding). This was read and discussed by the first two co-authors of the present paper, EEH and JS. Figure 1 highlights terms that either EEH, a molecular biologist (purple shading), or JS, an information theorist (yellow shading), found difficult to understand. Joining forces and explaining all terms to each other, they were able to understand the paragraph in depth.\n\nTerms that may not be clear to non-specialists in particular fields are highlighted in purple and yellow, corresponding to those causing problems for a molecular biologist and an information theorist, respectively. (Used under the Creative Commons Attribution 4.0 International License: http://creativecommons.org/licenses/by/4.0).\n\nAs the interdisciplinary field of DNA-storage evolves towards maturity, there will be an increasing requirement for researchers from different backgrounds to understand publications without having access to colleagues from unfamiliar subject areas. This can be achieved in part by including brief summaries, which may make use of our glossary document, in specialised sections of a publications such that they become accessible for researchers from all disciplines.\n\n\nConclusions\n\nWe promote the value of interdisciplinary, collaborative science to solve complex problems, including in our field of digital information storage in DNA which combines molecular biology, information theory and computer science. We note the problems that this approach can generate in communication within and between research teams, and propose to reduce these in the DNA-storage area by initiating a glossary and controlled vocabulary. These have been made available to the research community for reference and critique, and we invite contributions to extend their scope.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nEEH and JS are supported by the UK’s Biotechnology and Biological Sciences Research Council (BBSRC grants BB/L023741/1 and BB/L021994/1). NG is supported by the European Molecular Biology Laboratory.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank all participants at the IARPA meeting in Washington D.C. on 27–28 April 2016 (https://www.src.org/calendar/e006043/) for an interdisciplinary discussion, during which the need for a unified vocabulary to foster understanding within this new field was in evidence. We thank in particular Luis Ceze who chaired this discussion. This provided additional motivation for continuing and extending the glossary we had already put together, as reported during the meeting, and for writing this paper.\n\n\nReferences\n\nChurch GM, Gao Y, Kosuri S: Next-generation digital information storage in DNA. Science. 2012; 337(6102): 1628. PubMed Abstract \| Publisher Full Text\n\nGoldman N, Bertone P, Chen S, et al.: Towards practical, high-capacity, low-maintenance information storage in synthesized DNA. Nature. 2013; 494(7435): 77–80. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBornholt J, Lopez R, Carmean DM, et al.: A DNA-based archival storage system. In Proceedings of the Twenty-First International Conference on Architectural Support for Programming Languages and Operating Systems. ASPLOS ’16, New York, NY, USA, ACM. 2016; 44(2): 637–649. Publisher Full Text\n\nYazdi SM, Yuan Y, Ma J, et al.: A Rewritable, Random-Access DNA-Based Storage System. Sci Rep. 2015; 5: 14138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBuschmann T, Bystrykh LV: Levenshtein error-correcting barcodes for multiplexed DNA sequencing. BMC Bioinformatics. 2013; 14: 272. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLangmead B, Trapnell C, Pop M, et al.: Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3): R25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi H, Durbin R: Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics. 2009; 25(14): 1754–1760. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi R, Yu C, Li Y, et al.: SOAP2: an improved ultrafast tool for short read alignment. Bioinformatics. 2009; 25(15): 1966–1967. PubMed Abstract \| Publisher Full Text\n\nMardis E, McCombie WR: Library Quantification: Fluorometric Quantitation of Double-Stranded or Single-Stranded DNA Samples Using the Qubit System. Cold Spring Harb Protoc. 2017; 2017(6): pdb.prot094730. PubMed Abstract \| Publisher Full Text\n\nSchumacher B: Quantum coding. Phys Rev A. 1995; 51(4): 2738–2747. PubMed Abstract \| Publisher Full Text\n\nZhirnov V, Zadegan RM, Sandhu GS, et al.: Nucleic acid memory. Nat Mater. 2016; 15(4): 366–370. PubMed Abstract \| Publisher Full Text\n\nBrazma A, Hingamp P, Quackenbush J, et al.: Minimum information about a microarray experiment (MIAME)-toward standards for microarray data. Nat Genet. 2001; 29(4): 365–371. PubMed Abstract \| Publisher Full Text\n\nBustin SA, Benes V, Garson JA, et al.: The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem. 2009; 55(4): 611–622. PubMed Abstract \| Publisher Full Text\n\nLee JA, Spidlen J, Boyce K, et al.: MIFlowCyt: the minimum information about a Flow Cytometry Experiment. Cytometry A. 2008; 73(10): 926–930. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "29657", "date": "26 Feb 2018", "name": "Robert Grass", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by E. Hesketh addresses very important problems of our current scientific landscape, and the ongoing movement to more interdisciplinary approaches:\nCommunication between scientists in a team Peer Review\n\nThe authors discuss these two topics using a currently evolving research topic: the storage of digital information in DNA; but the addressed problems have a significantly broader applicability, as individual research topics spread over more and more scientific disciplines, and especially because data and computer sciences are having a major impact on science (and the corresponding high-level mathematics are currently not integrated into e.g. life-science curricula).\n\nFor the communication for scientists within a team, the authors present an excellent glossary of terms for the scientific fields involved in DNA data storage - and the development, and open publication/distribution of such glossaries would bring benefit to many interdisciplinary projects. Instead of a locally managed glossary (as proposed), are more open approach (e.g. as an open Wikipedia) would be even more beneficial and further motivate others to participate stronger in updating the glossary. Additionally, some referencing within the glossary would be additionally valuable - as often background in understanding an individual term is required. (as standard within Wikipedia). If the authors have good reasons for a non-public (i.e. wiki) approach, theses should be discussed in the article, if not, the generation of a corresponding wiki would be certainly highly appreciated by the research community.\n\nHowever, to completely solve the communication problems and misunderstandings in such projects, the authors touch a point of even higher importance: “misunderstandings can pass unnoticed”, so the question is what solutions are available to make team members aware of the danger of miscommunication and, implement sufficient effort for every individual in a given project to learn the details, wordings and backgrounds of the neighboring fields- the authors may want to further build on this observation, and potentially present approaches to ensure such awareness and openness (especially in teams involving specialists).\n\nThe second problem of interdisciplinary projects addressed is peer-review. The more detailed background of different scientific fields is required to judge the correctness of scientific work performed, the more difficult it is to find individuals as paper referees who cover all of this knowledge. A plain text summary, as presented by the authors as part of a solution is certainly a good start, but probably does not go far enough. In contrast to individuals working on an interdisciplinary project (as above), a journal referee does not have enough time to learn details and wordings of the other fields, and the review process gets somewhat superficial. A general understanding of the overall goals of a given paper (as per plain text summary) may help the referee to understand the article scope, but it will not help him to judge the scientific validity of the methods applied. The authors of the present manuscript somewhat touch on this, and a more explicit depiction of the problem may be valuable to a further discussion of future publishing/peer-review modes (e.g. post-publication review, open-review, various referees only refereeing part of articles).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "31971", "date": "29 Mar 2018", "name": "Jeffrey R. Sampson", "expertise": [ "Reviewer Expertise Nucleic acid synthesis and measurement technologies", "technology development and business strategy." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Hesketh et al., addresses the very important issue of facilitating productive communication among highly interdisciplinary teams. This impacts not only verbal communication among interdisciplinary members but also written communications in the form of simple messages and publications. It is also well noted that during peer review of publications, there is often lacking a single person with the necessary vocabulary and domain knowledge to fully understand, evaluate and communicate a review of the work. The method of Hesketh et al. will clearly aid in this important process. Importantly, they have developed a smart approach to the problem that can be applied more broadly to other interdisciplinary teams that require the integration of disparate fields of science and technology such as life sciences and engineering. For example, the synthetic biology community has experienced this issue as it has developed and evolved over the past 15 or so years.\nMore specifically, Hesketh et al. not only set a good structure and context that the interdisciplinary team developing the DNA as a digital information storage media face, but also provides some solutions to critical problems. The first is creating a glossary of terms so that all disciplines involved can communicate with a common and known set of terms. Second, they have put forward the use of a “controlled vocabulary” where terms that are particular to the emerging interdisciplinary field are defined so as to enable all members to communicate precisely and thus reduce confusion that often occurs when terms have multiple meanings and/or field dependent meanings. Perhaps most importantly, Hesketh et al., have built their approach as a “living document” where the vocabulary and common vocabulary can be continuously updated by the interdisciplinary community as the community grows and evolves.\n\nWith respect to any additional comments or edits, I offer that the authors consider adding “Chemistry Terminology” to their glossary with specific attention to the chemical synthesis of DNA since this is the current method for DNA synthesis. Such terms could include; phosphoramidite, cycle yield, coupling efficiency, de-block step, oxidation step.\nGiven the importance, clarity and potential for broad applicability, I strongly recommend the paper for indexing.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-39
https://f1000research.com/articles/7-24/v1	08 Jan 18	{ "type": "Research Article", "title": "Enhancing energy literacy in children using zn/cu/potato batteries", "authors": [ "Mark Polikovsky", "Avigdor Sharon", "Alexander Golberg", "Mark Polikovsky", "Avigdor Sharon" ], "abstract": "Background. The major challenges that prevent the wide-scale adoption of emerging personal clean energy production are unawareness and low self-confidence. We tested a hypothesis that a combination of a new technology and educational methods could lead to the increase in awareness of children to clean energy possibilities and to an increase in self-confidence in applying them. Methods. Here we report on a toolkit that combines low carbon, clean energy source, Zn/Cu/potato batteries, sufficient to power light-emitting diodes, with a non-formal education by experience program, based on case studies and hands-on experience with battery assembly for 6-11 years old children, led by trained 12-14 old youth leaders. Results. The results show that the education experience increased the awareness of the children to produce electricity at home from unconventional, yet available raw materials and their self-confidence in being able to do this (p=0.008). Conclusions. The developed toolkit supports environmental and energy literacy education through non-formal training, increasing awareness and self-confidence in children to actually apply this in their living environment to produce clean energy.", "keywords": [ "energy education", "batteries", "lighting", "household air pollution prevention", "bioelectricity" ], "content": "Introduction\n\nChildren are the decision-makers of the future and will bear the brunt of the energy-environment- climate crisis1–3. The USA Department of Energy Literacy guide stays:\n\nWithout a basic understanding of energy, energy sources, generation, use and conservation strategies, individuals and communities cannot make informed decisions on topics ranging from smart energy use at home and consumer choices to national and international energy policy4.\n\nA study of USA youth5 revealed that “students did not have a sound knowledge and understanding of basic scientific energy resources facts, issues related to energy sources and resources, general trends in the U.S. energy resource supply and use. The students also did not understand the impact of energy resource development and use on society and the environment”5. Similar results were observed in recent studies with New Zealand6 and Taiwanese7 youth, who mostly looked at energy in monetary terms and knew little of its environmental impacts. A UK study showed “that children derived more motivation to save energy from responsibility conferred by school activities than other (e.g. environmental) concerns, and some connected energy saving with dangers of using electricity (e.g. fire)”8.\n\nMost children learn about energy issues from parents6,8. Therefore, the energy literacy of young persons in low income countries is lower than in OECD countries because of the general population's unawareness of renewable energy potential and of energy impacts on health and the environment9. Developing education tools to increase awareness to the effect of energy production on health and environmental problems is a major challenge, required to make a change in this sector.\n\nOur long-term goal is to increase energy literacy among children by creating awareness to the hazards of toxic and polluting materials, the benefits of clean energy, and the possibilities of creating clean energy at home. Subsequently, to encourage learning and actual implementation, resulting in a behavior shift towards using clean energy on an on-going basis.\n\nTo achieve this goal it is important to provide not only the required knowledge, but also to develop interest, care and a perception of personal ability. These are all attributes of environmental literacy. Environmental literacy is the ability to understand the environmental systems that we live in, their importance and impact on us, as well as our own well-being requirements and to take the measures required to interact with the environment in a responsible fashion and to benefit from it while sustaining it for the long term10. It requires both cognitive and affective elements, encompassing knowledge, affects, skills and behavior, which are all important to develop responsible environmental behavior 10–12. As people develop their environmental literacy, they progress gradually from a basic, nominal level of their knowledge, emotions, skills and behavior to functional and operational levels (Figure 1a). Though each individual has a unique journey through the continuum of environmental literacy development, it is usual to go from initial awareness to developing personal concern and then better understanding and a perception of personal ability before deciding to take action10 (Figure 1b).\n\n(a) From rudimentary knowledge and budding awareness to responsible environmental behavior: the environmental literacy continuum. (b) The stages of developing environmental and energy-responsible literacy in education.\n\nGaining knowledge does not necessarily lead to developing pro-environmental attitude, care and values. Developing attitudes does not necessarily lead to taking action and to adopting environmentally responsible behavior13,14. Some triggers or specific ingredients of the learning experience and of life experience as a whole seem to be required to develop responsible environmental and energy-wise behavior. Such ingredients are personal experience and emotions, curiosity, sense of wonder and excitement15–20\n\nPrevious works suggested various scenarios to develop energy literacy through a standard curriculum in classes21. An alternative approach is an outdoor, non-formal education. Indeed, outdoor education, in a non-formal setting, was found to result in better behavioral outcomes in comparison to traditional classroom learning22. Several important attributes such as concentration, agility, emotional expressions, and communication were found to be higher in the outdoor education setting. It was reported that “Outdoor education influenced behavioral changes in a positive direction”22. Non-formal education at various settings provides new opportunities for learning and benefits for both the effective and cognitive axis of human behavior, inciting interest, positive attitudes and fond memories that persist over time23. The non-formal education, in an outdoors or community settings, is beneficial and synergistic with both school learning and family engagement24. Therefore, it is of an important significance in developing environmental and energy literacy and in nurturing responsible energy and environmental behavior in communities and families. Consequently, novel tools are needed to nurture this behavior.\n\nMore than two centuries ago, Luigi Galvani’s pioneering research of the electrical properties of biological tissues discovered “bioelectricity”. Inspired by those results, Alessandro Volta invented a device capable of producing electricity by the mere contact of conducting substances of different species”25. This device, the ‘Voltaic battery’, marked the birth of a new era in the development of modern physics and important changes in our lifestyle by using batteries. The key advantage of batteries is their ability to store and deliver portable, off-the-grid electricity.\n\nIn the application for lighting in low-income off-grid communities, batteries already allow for the transition from fuel to electric-based lighting and could reduce health risks26. In addition, by supporting the elimination of fuel-based light sources, they would also contribute to the reduction in greenhouse gas emissions27, weaning governments of the burden of fuel subsidies that often exceed expenditures on healthcare and, importantly, provide new jobs28,29. However, improperly disposed commercial batteries contaminate the environment with heavy metals such as mercury, lead, cadmium, and nickel. In addition, battery energy is the key cost component in all low-cost devices recently developed for low-income communities30–33.\n\nTo address the problems of availability, cost and environmental impact, we introduced vegetative batteries, based on Zn/Cu and potatoes with disintegrated cell walls. Preliminary cost analysis showed that a Zn/Cu-Potato battery is able to produce portable energy at ~7$/kWh, which is significantly lower than current available 1.5 Volt AA alkaline cell (~450$/kWh (retail)), flow (~600$/kwh)34, advanced lead-acid (900$/kWh)34, lithium ion (500$/kWh)34 or D cells (~49-84$/kWh)35. The basic battery cell design and resulting LED light (Figure 2) allows for reading a book in a completely dark room (Figure 2d). In comparison to kerosene lamps with lighting efficiency of 0.08-0.11 lm/watt, light-emitting diode (LED)-Zn/Cu-boiled potato battery provides 8.3-53.1 lm/watt. At the same time, cost of light is 0.13-0.85$/1000lm∙h, while for kerosene lamps it is 3.69-5.81 $/1000lm∙h35. This study was followed by a paper from Sri Lanka, which reported 500 hours operation LED with Zn/boiled plantain pith battery36. Yet, the rate of adaptation of new technologies in population is low. Low awareness and low self-confidence contribute to these low rates of technology adaptation37.\n\n(a) Basic components required to assemble a battery at home. Zinc and Copper plates (~5×10cm), wires, boiled potato, sealing tape, light emitting diode (LED). Test resistor and voltmeter are used for performance measurements. (b) Process for a single cell and complete battery assembly. Assembly description of four potato batteries to turn on one LED lamp (i) put 4 plates on a rigid substrate; (ii) add 4 boiled slices of potato, once cooled, on the metal plates, recommended to cover the metal plates with the potatoes, as much as possible; (iii) cover the potatoes with the Zn plates; (iv) seal the cells with a tape and connect the batteries with the cables - each metal should be connected to the closest battery to the second metal except to metal plates on the edges; (v) connect the not connected plates, to the LED lamp, the electric circle is closed; (vi) in a case everything connected right, the LED lamp will be turned on; (vii) to measure open circuit voltage, a voltmeter should be connected instead the lamp; (viii) to measure the current in the system, connect the 100ohm resistor instead of the LED and measure the voltage on it. (c) Schematic representation of a complete lighting device, powered by Zn/Cu/boiled potato battery. (d) Lighting device performance example for book page illumination in a completely dark room.\n\nThe goal of this work is to test the working hypothesis that Zn/Cu-boiled potato battery technology, taught using participatory outdoors educational methods, could lead to the increased awareness and self-confidence of young children in actually producing clean energy at home.\n\n\nMethods\n\n96 children aged 6–11 years and 11 youth guides 12–14 years old from Israel participated in the study in the “Teva HaSviva” summer camp in July-August 2015. Written informed consent was obtained from parents before the camp for their children’s participation in the study as part of their camp activities, according to the Israel law, by “Teva HaSviva”. The parents and the children were informed that the camp runs experimental educational programs in various branches of environmental education.\n\nWe developed an easy to use toolkit for light generation from LEDs. The important aspect of the potato battery toolkit (Figure 2a) is that it can be readily assembled at any home or any outdoor activity in the majority of the countries in the World. The basic elements of the battery are Zn and Cu electrodes, wires, LED, knife, boiled potatoes (Figure 2a). The steps for battery preparation, assembly, and testing appear in Figure 2b. Although in these studies we used conventional boiling, used by families to cook, alternative cleaner energy strategies, such as solar cooker can be used38.\n\nIn this work, we developed a “Make yourself a battery” toolkit to teach youth energy and environment issues with the potential to provide light and prevent household poisoning. We demonstrated the use of the kit as an educational tool in a pilot study with 96 children 6–11 years old. This kit can be used as an energy and environment educational technology for the outdoor educational activities. In addition, it can displace the kerosene lamp for reading in medium and low- income countries. The kit provides a low-cost technology that can serve the dual purposes of improving lighting efficiency and increasing environmental and energy literacy by removing unawareness and low self-confidence, which are major barriers for the introduction of new energy systems to low-income countries.\n\nDuring the activity, the children used boiled potatoes, Zn and Cu electrodes (5X10 cm), wires, resistors, voltmeter and LEDs, as described Figure 2a. The assembly protocol for each cell and the complete battery, which consists of several cells connected in series, is shown in Figure 2b and c. Figure 2 shows a step-by-step procedure for battery assembly and use to generate LED light sufficient for reading (Figure 2d)\n\nThe thickness of each galvanic cell was measured by ruler (1mm resolution). The working surface area was measured in digital images of each galvanic cell using ImageJ (ver 1.48r 18) program (NIH, ML). Open circuit voltage and the voltage on the 100ohm resistor were measured using a digital voltmeter (0.01 V resolution). Current flowing on the resistor was calculated by Ohm’s law as I=V/R. Power was calculated as P=I∙V. The current and power density were calculated by equations i=I/A (i=current density, A= area in cm2) and p=P/A, respectively39.\n\nTo increase energy literacy and the use of the “Make yourself a battery” toolkit, we designed the following pilot study. First, we further developed the technology of batteries based on Zn and boiled potatoes35, which can be easily used in low-income communities. Second, we assessed the pre-learning energy literacy of a group of children. Third, we performed a hands-on activity with the children who assembled and characterized the Zn/boiled potato batteries. Finally, we assessed the post-learning energy literacy of this group of children.\n\nTo enhance the environmental and energy literacy in youth we developed an outdoor activity that can teach children about energy conversion and light generation. The study was conducted at the “Teva Hasviva” summer camp at Tel-Aviv University, Israel in 2015. The camp takes place during every school vacation period that is at least 1 week long, throughout the year. The core themes of “Teva Hasviva” camps are sustainable living, care for nature and ecological literacy. The core mission of the camps is to foster environmental literacy, synergy with nature in and around the city, nature conservation and a healthy lifestyle. This is done in an enjoyable setting, learning by experience in a combined outdoors and university campus environment, having fun, leading activities with a practical contribution to the natural environment and to the community, research and discovery.\n\nThe study design is shown in Figure 3a. The summer activity is also a part of a 3-year qualification program for youth guides. For this study, we first trained young youth guides (12–14 years old), participating in a course during the same summer camp (N=11). The trained youth guides performed all the activities with children (6–11 years old, N=96), who participated in a 2-week summer camp. Additional activities for participating children included visiting natural open spaces near the city and conducting animals and plants surveys, building nesting boxes for birds, making creative artifacts from packaging materials to avoid dumping, learning to make healthy, natural food, plant ecological gardens, in a learning-by-doing approach (Supplementary File 1 and Figure 3b). Each part of the activities took 30–40 min.\n\n(a) The schematic description of the educational activity involving youth leader and children in the summer camp. (b) Teaching materials, lectures, and activity structure as performed during the camp.\n\nThe targets of the education activity were defined as follows:\n\n1. Theoretical introduction to energy in nature and society.\n\n2. Youth guides will learn how to teach and how to perform an experiment and then perform the experiment with the children.\n\n3. Children will learn about concepts of energy generation and its application to affluent and low-income countries problems.\n\n4. Children will learn of the actual production of electricity from Zn/Cu/potato batteries as an educational activity and as a tool for them to make their own battery to use.\n\n5. Development of energy education tool and protocol for rapid batteries assemblies for low- income remote communities.\n\nYouth guides, who participate in the three-year environmental leadership program of the camp, were instructed before their activity on the method to assemble the battery and measure the outputs of produced electricity. They practiced the theory of what they learned during the theoretical presentation. Moreover, they learned how to use the voltmeter to measure voltage and current and measure effective electrolyte surface area by digital photography. Each of the youth guides led a group of 5–8 children during the practical battery assembly activity. The assembly was done in a form of competition between groups. Youth guides taught the children about electricity production from batteries for lighting and showed them how to use the voltmeter. The winner was the team with maximum generated voltage. Youth guides were also responsible to collect measurements from the assembled batteries such as electrolyte area, number of connected-in-series cells, total voltage and current (Table S1 and Table S2).\n\nIn addition, to demonstrate to children that it is possible to provide human needs without damaging the environment, the complete life cycle of potatoes used in the process was shown (Figure S1). After the activity, all used potatoes were composted. No leftovers were thrown away. We explained that the composted potatoes could be used for fertilization. We explained that the metal plates, cables, and LED lamps could be reused for additional activates. This way we introduced the entire cradle-to-cradle lifecycle of all materials used. We described a process where we minimize any use of materials that could not be naturally recycled. In places where the teaching period is longer than a few weeks (as was in our case), and the places include area for potato growth and compost it can be useful to show the children the potato lifecycle before and after the activity, including cultivation of new potatoes using the compost from the previous ones. This activity can also be integrated with a field implementation of solar cookers38.\n\nA short pre-learning questionnaire and a matching short post-learning questionnaire were used. In both questionnaires, Likert-type questions (a 1–5 scale) for quantitative assessment of levels of knowledge, skills and attitudes and open text questions for qualitative analysis were used (Table 1, see Supplementary File 2 for detailed questionnaire structure). The questions addressed the major areas of environmental and energy literacy continuum with an emphasis on affects as a key to literacy development. Pre-learning questions were designed to find out the extent of awareness and the level of knowledge before the learning session took place. Post-learning questions were designed to find out what participants have learned and understood, what they remember and what was their attitude towards the information and insights from the learning session. All 96 children participating in the summer camp received the pre-learning questionnaire 4 days before the learning session. They received the post-learning questionnaire 4 days after the learning session. Both questionnaires were provided in a very casual, relaxed atmosphere during a time break at the orchard area where the children had lunch and rested. The questionnaire was kept short to make it easy for the children to reply to it despite distractions in the field. The results were then analyzed and compared.\n\nIn addition to descriptive statistics for the Likert-scale questions, Pearson correlation was computed to assess the relationship between answers to questions in the Likert-type questionnaire. Correlations are reported in APA style with the syntax r= Pearson’s correlation value, p=significance value. Student’s t-test (2-tailed, DF=is shown for each t-test result) was used to examine differences in variables before and after the learning session. All analysis was done with IBM SPSS Statistics ver 23 (IBM, NY).\n\nSome questions before and after the learning session were not exactly the same, therefore, no T value was calculated (before: “I think it will be interesting for me to learn about energy in nature”, after: “It was interesting for me to learn about energy in nature”).\n\n\nResults and discussion\n\nThe generated voltage, current, power, current density and power density from the assembled batteries are shown in Figure 4. The longest assembled by children battery consisted of 23 individual cells (Figure 4a). The largest generated open circuit voltage was 16.55 Volt (Figure 4b). The largest current was 2.8mA (Figure 4c), the largest generated power was 0.78mW (Figure 4d). The lowest battery galvanic apparent internal resistance was 1,424 Ohm (Figure 4e). The maximum generated current density was 77 µA cm-2 (Figure 4f) power density was 22 µW cm-2 (Figure 4g). The lowest galvanic internal resistivity was 42,320 Ohm∙cm (Figure 4h).\n\n(a) Number of cells in the final battery. (b) Open circuit voltage (OCV). (c) Current. (d) Power. (e) Galvanic apparent internal resistance (GAIR), which show how much energy is lost by battery internal resistance. (f) Current density. (g) Power density. (h) Internal resistivity.\n\nA short pre-learning questionnaire and a matching short post-learning questionnaire were used (Table 1. See Methods). In the pre-learning questionnaire, the children demonstrated a very high level of interest to learn about nature (4.38±0.92, on Likert-type 1–5 scale), as may be expected for children participating in a nature and environment summer camp. They also expressed a very high level of agreement that nature holds practical and useful value for us. This insight and the interest to learn were correlated (r=0.406, p=0.0).\n\nThe children were looking forward with much interest to learn and experience the creation of electricity from potatoes (4.23±1.23, on Likert-type 1–5 scale); less interest was expressed to learn about energy in nature in general (3.98±0.84). Although agreeing that we can create electricity by ourselves, they showed only an average level of confidence that they can do it. This infers a low confidence in their skills and locus of self-control. The interest to learn about creating electricity from potatoes was correlated to the interest to learn about energy in nature (r=0.398, p=0.0), but no correlation was found to the concept of creating electricity at home or to the level of confidence that they can do it (Table S3). On the other hand, there was a positive correlation between the perception that it is possible to create electricity on our own and the confidence that they can actually do it although they had no experience yet (r=0.375, p=0.0). Both findings show a positive interest and anticipation before the learning experience but little awareness and self-confidence in actually putting it to any practical use. We concluded that though they looked forward with positive anticipation to the learning session, they generally tended to expect it to be a curiosity with no practical implementation of actually producing electricity at home. Therefore, a major challenge for the learning activity for these children was to demonstrate and understand the practical implementation possibilities and to develop a perception of self-confidence in their capabilities to implement it. If the learning process would succeed with children in an affluent society in which power from the grid is constant and commonplace, it could potentially succeed even more in communities with no power from the grid. For future studies, we suggest to repeat this study with children in countries where power from the grid is scarce or non-existent and compare their attitude to the same process. It could be expected that they would be more aware from the start to the practical implementation yet this should be tested in a study.\n\nComparing the results before and after the learning session (Table 2 and Table 3), there was one significant difference - the perception that “I can create electricity at my home”. Since appreciation and interest to learn about nature was rather high to start with, a change in attitude was not expected to be significant. The challenge was in acquiring the skills and self-confidence or locus of control. A Student’s t-test analysis shows that there was a significant change in the perception that children can create electricity at home by themselves with such simple ingredients such as potatoes (t(178) = -1.74, p = 0.008), with higher scores after the learning session.\n\n“Which ways do we get or create electricity” top 7 replies.\n\n“How can we create electricity on our own”.\n\nThere was a marked correlation between the children’s perception that they can create electricity at their home and their thoughts that it was interesting for them to learn about energy in nature (r=0.328, p=0.02), interesting to learn to create electricity from potatoes (r=0.390, p=0.0) and that they enjoyed the learning session (r=0.430, p=0.0), Table S3.\n\nChildren that thought they could create electricity at home, were more comfortable to feel they can teach others what they learned (r=0.375, p=0.0). Again, so did children that thought it was interesting for them to learn about energy in nature (r=0.443, p=0.0), interesting to learn to create electricity from potatoes (r=0.617, p=0.0) and that they enjoyed the learning session (r=0.636, p=0.0). There was a high correlation between these insights (Table S3). These results show that achieving engagement and interest in the learning activity has a positive effect on developing perceptions of personal capabilities to actually create electricity at home from potatoes and to teach others.\n\nThese results for a learning session in the midst of an intensive summer camp show the potential of hands-on learning experience to set understanding and awareness and most of all, an appreciation of the personal capability to do it and make a change.\n\nThe purpose of the qualitative (open text) questions before the session was to find out the extent of awareness and the level of knowledge before the learning session took place (Table 2 and Table 3). The purpose of the questions after the session was to find out what they have learned and understood, what they remember and what was their attitude towards the information and insights from the learning session.\n\nWhen asked before the learning session: “How do we get or create electricity?” only 24% of participants mentioned the electric company or power stations, 14% mentioned solar energy, 20% either declined to answer or said they did not know, 13% talked about static electricity that they probably learned about in school or about batteries. Additional children mentioned non-relevant answers. Yet, the big surprise was that a remarkable 41% mentioned potato based battery as a source of electricity. It might be assumed that as they knew the questionnaire is related to the learning session, they thought that that was the reply we expected or wrote it for lack of any other thought.\n\nIn the post questionnaire, which was provided on the week following the activity, there was a marked difference in replies to the same questions. When asked this time how do we get or create electricity, 23% mentioned solar energy and other renewable energy sources. Potatoes reined high with 52% of replies. Only 19% mentioned the electric company or power stations, and 15% declined to answer or said they did not know. These results show that a week after the learning session there was a high acclamation to the use of renewable energy sources for electricity and of the possible use of potato batteries as was experienced at the learning session.\n\nConsequently, it appears that before the learning activity only 33% of the children mentioned valid sources of electricity at home for daily use. After the activity, there was a larger awareness to solar energy and other renewable sources. Non-relevant sources, such as lightning and static energy, that appeared on replies before the learning activity (13% of the children) were dropped after the activity.\n\nWhen asked what they know about the possibility of creating electricity at home, 60% mentioned again potatoes, but an additional 12% knew to explain exactly how it is done. Another 5% talked about using lemon or vinegar and 8% talked about renewable energy sources. 24% either declined to answer or said they did not know, 8% talked about static electricity or batteries, and 3% said explicitly that it could not be done.\n\nBefore the learning session, 18% of participants knew about ways to create electricity at home, 23% had no idea, and 41% just wrote “potatoes”. After the session, 15% of the children provided a detailed account of how to make a battery while an additional 18% provided a partial explanation. Additional 6% of participants tried to explain, but provided very wrong explanations. 21% declined to reply while 29% wrote they do “not know” or “don’t remember”. 13% knew to explain how the batteries work while 12% tried to but provided inaccurate explanations.\n\nThese levels of 33% being able to reproduce at least partially the battery-making process and 13% being able to repeat even more complex scientific or technological explanations show a very promising pilot study for the large scale adaptation of new energy systems through learning. These children were at a summer camp where they are playing and having fun most of the time and experience many workshops and learning activities as they go from one station to another. So they were surrounded with distractions and leisure activities. In these circumstances, it was notable to achieve such levels of attention and actual learning. A future study could evaluate the effect of repeated learning session or a more extended learning program to see if a larger percentage of renewable energy literacy, awareness and perception of personal capabilities is achieved.\n\nBeyond these accounts of personal learning, the children were asked if they think now that Zn/Cu/boiled potato batteries can provide cheap electricity in a simple, easy-to-make way. 54% agreed that yes, it can. 6% said it might be possible but in low volumes, not enough for real consumption. 5% said that it is impossible.\n\nWhen asked so how can this be achieved, 62% did not reply to this question or said they did not know or did not remember. 31% said they could do it, mostly with the assistance of professional adults, such as a teacher or technician, or with the assistance of parents and family members. The break-down of these replies can be seen in Table 4.\n\nN is the number of children who replied.\n\n\nConclusions\n\nAlthough renewable energy sources are continuously developed, their penetration and adaptation rate by rural population in developing countries is low. These low adaptation rates are associated with unawareness for an alternative to traditional methods solutions and low self-confidence to adopt the new methods. In this study, we developed a toolkit based on Zn/Cu/boiled potatoes batteries and non-formal teaching strategy that could enhance energy literacy and potentially displace the kerosene used for lighting in rural areas. Our pilot study on 96 children from an Israel summer camp, trained by youth leaders, showed that informal renewable energy education increases the awareness and self-confidence for battery assembly and electricity generation at home using simple available materials.\n\n\nEthical statement\n\nThe study was conducted according to the research guidelines of the Chief Scientist Office of the Ministry of Education. The educational institution, Teva HaSviva camp, approved of the research plan and informed all parents/guardians about the exact activities that the children would participate in, including interviewing the children, filming and distributing the information online for professional and educational purposes. We received written informed consent from the parents/guardians for the children to participate in the study as a part of their summer camp activities. The research involved only the use of non-sensitive, completely anonymous educational tests, using anonymous questionnaires and interview procedures that did not induce any undue psychological stress or anxiety. This study was done within normal education requirements in Teva HaSviva and to the best of our knowledge is exempt from ethical approval, as per Israel law, which follows Helsinki convention regulation.\n\n\nData availability\n\nDataset 1: Questionnaire Raw Data Kids - Pre Learning. DOI, 10.5256/f1000research.13228.d18898940\n\nDataset 2: Questionnaire Raw Data Kids - Post Learning. DOI, 10.5256/f1000research.13228.d18899041\n\nDataset 3: Raw data for boiled potato batteries performance. DOI, 10.5256/f1000research.13228.d18899142", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors acknowledge the cooperation and assistance of the “Teva HaSviva” summer camp 2015 team and all the children who participated in this study.\n\n\nSupplementary material\n\n>Supplementary File 1. Teaching materials for renewable energy learning session.\n\nClick here to access the data.\n\n>Supplementary File 2. Pre and post learning questionnaires.\n\nClick here to access the data.\n\nTable S1. Zn/boiled potato battery characterization table as filled by children.\n\nClick here to access the data.\n\nTable S2. Electricity generation competition results. Measurements and calculations made by the children during competition. Va- voltage on external 100 ohm resistor connected in series with the potato battery. OCV- open circuit voltage.\n\nClick here to access the data.\n\nTable S3. Pre and post learning questionnaires analysis.\n\nClick here to access the data.\n\nFigure S1. Digital images and illustrations of the potato lifecycle during the activity with 96 children. (a) Two bags of potatoes (about 4 kg each) were used during the activity. (b) The potatoes were cut into pieces. (c) The central part of each potato cut with 1–2 cm intervals, and divided between the central part and the edges. (d) The potatoes were boiled during about an hour. (e) The boiled potato were divided into a mash potato and for slices used the activity purposes ⅓ and ⅔ respectively. (f) All used potatoes after the activity with the children were composted.\n\nClick here to access the data.\n\n\nReferences\n\nZyadin A, Puhakka A, Ahponen P, et al.: School students’ knowledge, perceptions, and attitudes toward renewable energy in Jordan. Renew Energy. 2012; 45: 78–85. Publisher Full Text\n\nBoylan C: Exploring elementary students’ understanding of energy and climate change. Int Electron J Elem Educ. 2008; 1(1). Reference Source\n\nLambert JL, Lindgren J, Bleicher R: Assessing Elementary Science Methods Students’ Understanding About Global Climate Change. Int J Sci Educ. 2012; 34(8): 1167–87. Publisher Full Text\n\nDOE: Energy Literacy: Essential Principals and Fndamental Concepts for Energy Eduction. 2014. Reference Source\n\nBodzin AM: What Do Eighth Grade Students Know About Energy Resources? In: NARST 2011 Annual International Conference. 2011. Reference Source\n\nAguirre-Bielschowsky I, Lawson R, Stephenson J, et al.: Energy literacy and agency of New Zealand children. Environ Educ Res. 2017; 23(6): 832–854. Publisher Full Text\n\nChen KL, Liu SY, Chen PH: Assessing multidimensional energy literacy of secondary students using contextualized assessment. Int J Environ Sci Educ. 2015; 10(2): 201–18. 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J Environ Educ. 2007; 39(1): 45–59. Publisher Full Text\n\nNegev M, Sagy G, Garb Y, et al.: Evaluating the Environmental Literacy of Israeli Elementary and High School Students. J Environ Educ. 2008; 39(2): 3–20. Publisher Full Text\n\nCarson R: The Sense of Wonder. 1950.\n\nReynolds HL Ed.: Teaching Environmental Literacy: Across Campus and Across the Curriculum. Indiana University Press; 2010. Reference Source\n\nOrr DW: Ecological Literacy. In: Ecological Literacy - Education and the Transition to a Postmodern World. State University of New York Press; 1992; 85–95. Reference Source\n\nWilson AR: Fostering a sense of wonder during the early childhood days. Greyden books; 1993. Reference Source\n\nWilson RA: Starting Early: Environmental Education during the Early Childhood Years. ERIC Dig.1996. Reference Source\n\nCobb E: The ecology of imagination in childhood. New York: Columbia University Press; 1977. Reference Source\n\nKirby SD, Guin AG, Chilcote AG: Creating the next generation of residential energy stewards using the energy transformation curriculum. Hous Soc. 2015; 42(3): 250–8. Publisher Full Text\n\nFiskum TA, Jacobsen K: Relation Between the School Environment and the Children’s Behaviour. Open Educ J. 2012; 5: 39–51. Publisher Full Text\n\nEshach H: Bridging in-school and out-of-school learning: Formal, non-formal, and informal education. J Sci Educ Technol. 2007; 16(2): 171–90. Publisher Full Text\n\nHenderson AT, Mapp KL: A New Wave of Evidence: The Impact of School, Family, and Community Connections on Student Achievement. Annual Synthesis 2002. National Center for Family and Community Connections with Schools. 2002. Reference Source\n\nVolta A: On the Electricity Excited by the Mere Contact of Conducting Substances of Different Kinds. In a Letter from Mr. Alexander Volta, F. R. S. Professor of Natural Philosophy in the University of Pavia, to the Rt. Hon. Sir Joseph Banks, Bart. K. B. P. R. S. Philos Trans R Soc London. 1800; 90: 403–31, [cited 2016 Oct 13]. Publisher Full Text\n\nKimemia D, Vermaak C, Pachauri S, et al.: Burns, scalds and poisonings from household energy use in South Africa: Are the energy poor at greater risk? Energy Sustain Dev. 2014; 18(1): 1–8. Publisher Full Text\n\nTedsen E: Black Carbon Emissions from Kerosene Lamps. Berlin; 2013. Reference Source\n\nMills E: Job creation and energy savings through a transition to modern off-grid lighting. Energy Sustain Dev. 2016; 33: 155–66. Publisher Full Text\n\nMills E: Light for Life: Identifying and Reducing the Health and Safety Impacts of Fuel-based Lighting. UNEP. 2014. Reference Source\n\nWere FH, Kamau GN, Shiundu PM, et al.: Air and blood lead levels in lead acid battery recycling and manufacturing plants in Kenya. J Occup Environ Hyg. 2012; 9(5): 340–4. PubMed Abstract \| Publisher Full Text\n\nMeyer PA, Brown MJ, Falk H: Global approach to reducing lead exposure and poisoning. Mutat Res. 2008; 659(1–2): 166–75. PubMed Abstract \| Publisher Full Text\n\nSuplido ML, Ong CN: Lead exposure among small-scale battery recyclers, automobile radiator mechanics, and their children in Manila, the Philippines. Environ Res. 2000; 82(3): 231–8. PubMed Abstract \| Publisher Full Text\n\nGottesfeld P, Pokhrel AK: Review: Lead exposure in battery manufacturing and recycling in developing countries and among children in nearby communities. J Occup Environ Hyg. 2011; 8(9): 520–32. PubMed Abstract \| Publisher Full Text\n\nIFC: Energy Storage Trends and Opportunities in Emerging Markets [Internet]. 2017. Reference Source\n\nGolberg A, Rabinowitch HD, Rubinsky B: Zn/Cu-vegetative batteries, bioelectrical characterizations, and primary cost analyses. J Renew Sustain Energy. 2010; 2(3): 033103. Publisher Full Text\n\nJayashantha N, Jayasuriya K, Wijesundera R: Biodegradable Plantain Pith for Galvanic Cells. In: Proc 28th Tech Sess Inst Phys, Sri Lanka. 2012; 28: 92–7. Publisher Full Text\n\nMuddassir M, Noor MA, Ahmed A, et al.: Awareness and adoption level of fish farmers regarding recommended fish farming practices in Hafizabad, Pakistan. J Saudi Soc Agric Sci. 2016. Publisher Full Text\n\nCuce E, Cuce PM: A comprehensive review on solar cookers. Appl Energy. 2013; 102: 1399–421. Publisher Full Text\n\nTiwari G, Saxena DRK: The Regenerative Energy Suspension System. Int J Sci Eng Res. 2015; 6(4): 1249. Reference Source\n\nPolikovsky M, Sharon A, Golberg A: Dataset 1 in: Enhancing energy literacy in children using zn/cu/potato batteries. F1000Research. 2017. Data Source\n\nPolikovsky M, Sharon A, Golberg A: Dataset 2 in: Enhancing energy literacy in children using zn/cu/potato batteries. F1000Research. 2017. Data Source\n\nPolikovsky M, Sharon A, Golberg A: Dataset 3 in: Enhancing energy literacy in children using zn/cu/potato batteries. F1000Research. 2017. Data Source" }	[ { "id": "29584", "date": "14 May 2018", "name": "Srikanth Mutnuri", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper is well written. However I am not sure how the potato experiment can increase the awareness of children about clean energy possibilities, and increase their self-confidence in applying them. This experiment can certainly give knowledge about how electricity can be generated. I am not sure about it as clean energy possibilities. I feel this can be highlighted in the paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "42920", "date": "28 Jan 2019", "name": "Ikerne Aguirre Bielschowsky", "expertise": [ "Reviewer Expertise Household energy consumption", "energy literacy", "environmental education", "environmental consciousness and practices", "and cultural comparisons." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI found this article interesting, useful for educators, well written and structured, and easy to follow. All of the raw data are made available, and the paper provides enough information to replicate the activity. The ideas presented here are innovative, and could have practical implications when applied in a low-income, off-the-grid context – indeed, I very much look forward to the follow-up study focusing on this topic. Before moving on, however, there are a few points that need to be clarified.\nMajor comments\nOverall, I think that the framing of the study needs to be more explicit on what the authors wish to achieve and, crucially, what they hope to achieve here vs in a future study building on the current results. The abstract and introduction suggest a focus on developing the children’s awareness of energy sources and their impact on society and the environment, as well as changing their energy behaviour. Yet the project itself mostly deals with the practical and scientific side of making electricity from potatoes. If the children were involved in discussions on (the impact of) current energy sources, and/or what difference using potatoes could actually make, please elaborate on what specific topics were discussed, how they were discussed, and the final understanding obtained by the children. I think the paper ought to be clear throughout on what are the secondary aims (apart from building self-confidence) E.g. to (i) increase children’s scientific knowledge on the process of electricity production; (ii) serve as a pilot study for a bigger research programme, which intends to improve living conditions in off-grid areas; and/or (iii) develop children’s energy literacy. All three are worthwhile, and indeed all of them are mentioned here; however, not all of them are properly discussed, especially the energy literacy part. For example, the children are seemingly meant to realise the connection between generating electricity from potatoes and the wider socio-environmental implications of energy consumption and production. Understanding this complicated relationship likely would require different or additional learning experiences and follow-up evaluations. According to the results (Table 2), most children think that electricity in general is generated from potatoes (both before and after the activity). This statement somewhat worries me, as it seems to reveal a misunderstanding about how electricity is actually produced in our society. Is there any indication that the children understand the broader context? It would be helpful to have an idea of the characteristics of the participating children. How many boys vs girls participated? Are they mostly the children of academics, or do they come from underprivileged neighborhoods? What is their general socioeconomic background? Also, there ought to be some information on their previous experience. Is energy education compulsory in primary schools in Israel? Would the children have had previous experience doing experiments involving electricity, conductivity, etc.?\n\nMinor comments\nIt is important to acknowledge in the introduction (e.g. bottom of p.2) that energy-related behaviours depend on external factors as well as internal/psychological ones, e.g. material culture and social norm. The latter are especially important when working with children. Educators thinking of running this project with their students might benefit from an idea of how much a complete kit costs. I think you mean technology “adoption”, rather than “adaptation”. The section on “Qualitative assessment of knowledge and attitude” is somewhat repetitive, as it mostly recalls information already presented in the tables. I think it could be significantly shortened by discussing only the main trends. Also, I wonder whether the qualitative aspect of this section could be enhanced via some quotes, and a more in-depth discussion of the participants’ opinions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "43040", "date": "11 Feb 2019", "name": "Sue Wake", "expertise": [ "Reviewer Expertise Children and youth environments including participatory theory and children's inclusion in design decisions. Children's rights and environmental education." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nYour paper is interesting and relevant, but there are a number of areas that should be attended to, in order to strengthen its content readability and therefore its research value. I outline these here, in the order of reading:\n1. You should state upfront that this is a pilot study and you are using child participants from affluent backgrounds, with the assumption that the results should be more valuable if applied to children from poorer backgrounds. You should provide some evidence of why you have this assumption - e.g. academic studies. Currently it is confusing as you introduce the idea of the pilot study further into the paper, and, even later, you admit that your subjects are from wealthy backgrounds and you recommend repeating the project with poorer children, since this could/should yield more positive results.\n2. In your introduction you should more obviously credit the 2 models in Figs 1a and b. The way it is currently presented makes them seem like your own.\n3. The paragraph in your intro starting 'Previous works...' is inelegantly written and not well linked to the topic of this paper. I recommend you reduce its length and more strongly connect it to your project in terms of your chosen focus (i.e. informal outside education).\n4. It feels that there is a lost opportunity in this paper with regards making more of the youth leader role. This is an excellent example of 'peer learning' and reference to the literature on this (under children's participation) would give greater depth to your research and also extend the learning potential of your project. An example reference is Harry Shier's 'Pathways to participation' revisited: Learning from Nicaragua's child coffee workers1.\n\n5. The second to last para of your intro, beginning 'At the same time ... the cost of light...' What do you mean by the cost of light? You then write 'This study was followed...' This sentence needs clarifying and more detail provided. For example, you might say 'A similar study from Sri Lanka found ... due to lack of awareness and poor self-confidence'. The word 'populations' should have the 's' removed.\n\n6. In general you don't have enough references to the qualitative aspects of your research. More background on this will strengthen your argument considerably.\n7. Your sections on 'Battery Performance Evaluation' and 'Electrical Production' sit very awkwardly in relation to the context of your research. I question what is the relevance of these sections? If it is important to your paper to produce these figures, then you need to put them into context and explain why they are relevant to your paper.\n8. The information about the youth guides on P.5 is a little ambiguous as to whether the whole section just refers to them, or also to the other child participants. Perhaps give this a section heading e.g. 'Youth Guide Training'.\n9. Also P.5 - I think you mean 'fertiliser', not 'fertilisation'.\n10. 'Results & Discussion' - the sentence about the longest battery should be changed to 'longest battery assembled by children' - to make sense.\n11. There are a number of places where the English is not quite correct and the whole paper needs close editing for this. For example, where 'can' should be replaced by 'could'.\n\n12. Is Fig 4 really of value to your paper? I am not convinced it is.\nOverall, while your paper about the pilot study has some valuable outcomes, there are some significant issues with the number of assumptions you make, without evidence to support your rationale. Strong evidence of the benefits to children's education about alternative power sources and the viability of this method as a way of generating bio-energy (e.g. a whole lot of cooked potatoes presents a space and hygiene problem) seem to be rather absent from your paper. The observation that the 41% of participants that mentioned potatoes as an energy source, probably guessed this answer from inferences already made to them, sort of sums up the inconclusiveness of your results. This is reflected in your very brief conclusion. The paper needs to gain more substance through reducing some of the current content (as suggested) to allow space to focus more strongly and clearly on the background to children's learning by this method, linked through to your results. Currently there are too many loose ends. I also recommend that if you do any follow-up research, that you include the voice of the youth leaders and questions asked of the child and youth participants about how this way of learning compared to learning from adults.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "4627", "date": "07 May 2019", "name": "Avigdor Sharon", "role": "Author Response", "response": "Thank you very much for your remarkable comments!Here are replies, numbered according to comments. Thank you for this insight. We will review this text and update accordingly. The models in Figs 1a and b are indeed our own original charts. We will update the text to make it explicit Thank you for this insight. We will review this text and update accordingly. Indeed the youth leaders and peer learning were both major principles in the learning program. However, the sample of youth leaders was not large enough to be statistically significant. Therefore it was dropped. We will review and expand on this important topic. Thank you for the link. Thank you for this insight. We will review this text and update accordingly. Thank you for this insight. We will review this text and update accordingly. Thank you for this insight. We will review this text and update accordingly. This section refers to both the youth guides and to the children. But we will review if we can separate it to a youth guide training section and to a children education section. The purpose was indeed to refer to ‘use for fertilization’. Will expand it to explain the process of composting. We will update accordingly the grammar of this sentence. We will look for these places and update accordingly the grammar. Thank you again for this insight. We will review and update accordingly. We aimed indeed to include the voice of the youth leaders and find this feedback from them but the number of comments we received was not enough to justify inclusion. This would be a major issue to study in subsequent research" } ] } ]	1	https://f1000research.com/articles/7-24
https://f1000research.com/articles/6-2034/v1	21 Nov 17	{ "type": "Method Article", "title": "Identification of organic pigments in tattoo inks and permanent make-ups using MALDI-TOF mass spectrometry", "authors": [ "Markus Niederer", "Urs Hauri", "Lydia Kroll", "Christopher Hohl", "Urs Hauri", "Lydia Kroll", "Christopher Hohl" ], "abstract": "Nowadays, about 12% of the European and 20% of the US population are tattooed. Rising concerns regarding consumer safety, led to legal restrictions on tattoo inks and permanent make-up (PMU) inks. Restrictions also include bans on certain hazardous colourants. Both ink types use organic pigments for colour-giving, plus inorganic pigments for white and black and colour tones. Pigments are only sparingly soluble in common solvents and occur as suspended particles in the ink matrix. Their detection and identification therefore pose a major challenge for laboratories involved in monitoring the legal compliance of tattoo inks and PMUs. We overcame this challenge by developing a matrix-assisted laser desorption ionisation time-of-flight mass spectrometry method, which included an easy sample clean up. The method proved to be capable of detecting and identifying organic pigments in almost all of the tested ink samples. Method validation and routine deployment during market surveys showed the method to be fit for purpose. Pigment screening of 396 tattoo inks and 55 PMUs taken from the Swiss market between 2009 and 2017 lead to the following conclusions: Pigment variety is much greater in tattoo inks (18) than in PMUs (10); four prohibited pigments (Pigment Green 7, Pigment Red 122, Pigment Violet 19 and 23) were found in both ink types; for PMUs, these four pigments made up 12% of the pigment findings, compared to 32% for tattoo inks. Therefore, legal compliance of PMUs was at a higher level. A comparison of pigments found with those declared on tattoo ink labels clearly showed that banned pigments are rarely declared, but rather masked by listing not present legal pigments and label forging; therefore, highlighting the urgency of widespread market controls.", "keywords": [ "tattoo inks", "PMUs", "colour pigments", "MALDI-TOF" ], "content": "Introduction\n\nOver the last two decades, tattoos have become increasingly popular among younger people, with estimates stating that 12% of the European and more than 20% of the US population are tattooed1. These figures, the widespread on-line sales of tattoo related products, and the invasive techniques used during tattooing - where inks are injected into the skin’s dermis - have led to serious concerns on the safety of tattoo inks and permanent make-ups (PMUs). This finally compelled several European nations to initiate specific legal regulations, which came into effect during the last ten years and are all based on the European Resolution (ResAP 2008)2,3. For colourants, namely pigments, being the ingredients, which give tattoo inks and PMUs the desired effect, regulations are summarised as follows: a ban on colourants that can form aromatic amines under reductive cleavage, a negative list for specific colourants and a ban for colourants with restricted use in cosmetics. Despite this welcome development, safety awareness in this field is still not adequate compared to cosmetics, which have been subjected to stringent regulations for decades, even though their application forms are solely non-invasive. Regulations for cosmetics also require manufacturers to evaluate the consumer safety of their cosmetic products and ingredients. In contrast to this, none of the ingredients used in tattoo inks have ever been tested to ensure their health safety when injected into the skin. Furthermore, the generalised assumption that pigments as insoluble colourants pose no health risk, does not hold true in the dermis. Pigment fading and transport of organic pigments from the skin to regional lymph nodes has been well documented for tattoos4,5. Studies have reported that diarylide pigments degrade under sunlight forming a variety of products, some of which are known to be toxic or carcinogenic6. This finding has yet to be incorporated into regulations.\n\nPigments are at best only sparingly soluble in common solvents, which narrow the choice of suitable analytical techniques. So far the identification of tattoo pigments in inks has mainly been carried out using Raman as well as Fourier transform infrared (FT-IR) spectroscopy7,8. Raman spectroscopy has also been used for matching the profile of tattoo inks with dermatome shave biopsies from patients with allergic reactions9. Another recent paper describes the successful use of pyrolysis gas chromatography (py-GC/MS)10. While FT-IR and Raman Spectroscopy are very suitable for the identification of single pigments, their successful use for pigment mixtures, often present in tattoo inks, has yet to be proved. Matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF–MS) has been reported for identifying pigments in automotive coatings11 or art work8,12–14. In this article, we present a validated MALDI-TOF-MS approach for the identification of pigments in tattoo inks and PMUs. Repeated market surveys in Switzerland showed the method to be fit for purpose.\n\n\nMethods\n\nAs reference materials, more than 150 individual pigments from different producers (mainly BASF (Basel, Switzerland), Clariant (Muttenz, Switzerland) supplied by Omya (Oftringen, Switzerland), Kremer Pigmente (Aichstetten, Germany) and Sigma–Aldrich (Buchs, Switzerland)) were collected over the past years. Mass calibration was performed with the following dyes all from Sigma-Aldrich: Crystal Violet (C.I. 42555), Basic Blue 7 (C.I. 42595), Ethyl violet (C.I. 42600), Basic Blue 11 (C.I. 44040), Basic red 2 (C.I. 50240), Basic Blue 17 (C.I. 52040). MALDI matrix 2,5-Dihydroxybenzoic acid (DHB, 99%) was purchased from Sigma–Aldrich. Ethanol (EtOH) and methanol (MeOH), both analytical grades, were from J.T. Baker (Center Valley, USA).\n\nStock suspensions and/or solutions of reference pigments in MeOH (1 mg mL-1) were individually prepared. For mass calibration, a mixture containing six pigments at levels of 50 μg mL-1 for C.I. 42555, C.I. 42595 and C.I. 42600, of 250 μg mL-1 for C.I. 50240 and C.I. 52040 and of 100 μg mL-1 for C.I. 44040 was prepared by mixing homogenous aliquots of each stock suspension and diluting with MeOH.\n\nTattoo inks and PMUs were taken from the Swiss market over the last eight years. An overview of the analysed samples is given in Table 1.\n\nInk samples were shaken intensely by hand for about one minute. Then, about 25 µL (20–30 mg) of the suspension or 20 mg of PMUs were weighed into a tared 2 mL-tube, mixed with 1 mL of EtOH, sonicated (5 min, 25°C) and centrifuged (5 min, 15000 rpm). The supernatant was removed and the residue containing the colour pigments was diluted with 1 mL of MeOH. After vortexing for some seconds, 1 µL duplicates of the homogenous suspension were immediately spotted onto a steel target plate, air dried at room temperature and used for LDI-TOF-MS.\n\n20 µL of the methanolic ink suspension were transferred to a 96-Well reaction plate (MicroAmp Optical, Applied Biosystems) and mixed with 10 – 50 µL of the appropriate reference pigment suspension (10 mg mL-1 MeOH). One microliter of the homogenous suspension was used for LDI-TOF-MS.\n\n20 µL of the methanolic ink suspension were transferred to the reaction plate, mixed with the same volume of DHB (10 mg mL-1 MeOH) and 1 µL of the homogenous suspension was used for MALDI-TOF-MS.\n\nPigment mass spectra were obtained using a MALDI-TOF Mass Spectrometry Axima™ Confidence machine (Shimadzu-Biotech Corp., Kyoto, Japan) with detection in the reflectron mode with pulsed extraction (optimised at 450 Da) at a N2-laser frequency of 50 Hz and within a mass range from 50 to 2000 Da. The transmission of the laser power was in the range of 60 – 100 units (33 – 55%). The target plate was scanned by the laser (diameter of 30 μm) in the regular rectangular mode and serpentine style (1000 x 1000 μm, spacing 166.666 μm and 49 points). A minimum of 20 laser shots was accumulated per profile and for each sample a total of 50 mass profiles was averaged and processed using Launchpad™ version 2.9.3 software (Shimadzu-Biotech Corp., Kyoto, Japan). This software was also used for peak processing of all raw spectra with the following settings: the advanced scenario was chosen from the parent peak clean up menu, peak width was set to 3 channels, smoothing filter width to 2 channels, baseline filter width to 10 channels, and the threshold apex was chosen as the peak detection method. The threshold apex peak detection was set as a dynamic type and the offset was set to 0.300 mV with a response factor of 1.0. The processed spectra were exported as peak lists with m/z values for each peak and signal intensity in the ASCII format. Calibration was conducted for each target plate using spectra of the reference standard mixture consisting of the following exact masses: 271.1137, 315.1609, 372.2439, 422.2596, 456.3378 and 478.3222 amu.\n\nExported ASCII files from MALDI-TOF-MS were converted to NIST software compatible peak lists with a mass resolution of 1 D (.msp), and the identification of pigments was carried out with our home-made mass spectra library using the software of NIST 2.0 (Standard Reference Data Program of the National Institute of Standards and Technology, USA).\n\nMore or less soluble colourants detected by MALDI were occasionally confirmed by high performance liquid chromatography (HPLC). Approximately 5 mg of tattoo or PMU samples were extracted with different solvents, starting with N,N-Dimethylformamide, 1-Chloronaphthalene and N-Methylpyrrolidone, 1 mL each. Coloured extracts were centrifuged at 12’000 g, filtered with 0.45 µm PTFE syringe filters and analysed with Ion Pair Reversed Phase HPLC with Ultraviolet Diode Array (UV/DAD) detection under the following conditions: Kromasil-column C18, 5 µm, 150 x 2 mm (30°C); eluent A: aqueous solution of dodecyltrimethylammonium bromide (3 g/L) and ammonium bromide (1 g/L), eluent B: ethanolic solution of dodecyltrimethylammonium bromide (3 g/L) and ammonium bromide (1 g/L); run time = 30 minutes; flow rate = 0.35 mL/min; gradient conditions: 0 min 45% eluent B, 2 min 55% eluent B, 10 min 65% eluent B, 20 min 100% eluent B, 24 min 100% eluent B, 24.1 min 45% eluent B.\n\n\nResults and discussion\n\nOver the last eight years, more than 150 individual colour pigments and about 450 commercial tattoo ink or PMU samples from market surveys were subjected to LDI- or MALDI-TOF MS analyses. Mass profiles were recorded either from pigments of different producers in order to build a home-made reference spectrum library or from samples taken during market surveys. From these former investigations we derived the following conclusions: MALDI-TOF MS is an excellent and rapid method for the detection and identification of organic colour pigments, but not for inorganic ones. For the most part of the analysed pigments, the ionisation can be performed without added matrix (LDI-TOF), because the pigments themselves function as chromophores absorbing the laser beam14.\n\nAfter clean-up, samples were screened with LDI-TOF MS using the NIST program with our pigment library for identification. Tentatively identified pigments were then confirmed by comparing the high-resolution mass spectra of the native and the reference spiked sample. As a further check, the resulting colour of found pigments had to give the sample colour. Whenever the results of the analysis did not match with the colour of the samples, the ionisation was optimised by changing the laser power and/or adding DHB as a matrix to the sample.\n\nModern tattoo inks not only contain pigments but also binders, solvents, surfactants, preservatives and thickening agents15. Surfactants adsorb to the surface of pigment agglomerates and decrease the surface tension of the solvent. This eliminates residing air bubbles and improves particle coating with other additives. Adsorbed surfactants, however, can interfere with analysis; spiking C.I. 74265 (pigment green 36) with the non-ionic tenside Triton X-100 leads to a nearly complete suppression of mass signals (Figure 1). This problem was met by washing samples with EtOH before LDI/MALDI, which proved to be effective in most cases16.\n\nHigh resolution mass spectra of Pigment Green 36 (C.I. 74265) without any discrimination effect (A) and affected by the surfactant Triton X-100 (B).\n\nWith no commercial library of pigments available, we made our own library by exporting ASCII files of the mass spectra of analysed reference pigments to NIST compatible unit resolution mass peak lists. We preferred the NIST software for screening samples because its reversed match algorithm (R. Match) allows the simultaneous identification of several pigments in mixtures. This advantage is demonstrated for a red tattoo ink in Figure 2. Although the three existing pigments have overlapping ion clusters, all could be identified by their characteristic ions: C.I. 73915 (P.R. 122) with the ions [M+H]+ (m/z 341) and the sodium adduct [M+Na]+ (m/z 363), C.I. 21110 (P.O. 13) with the molecular ion [M+H]+ (m/z 623) and the two fragment ion clusters at m/z 437 and m/z 251 and C.I. 12475 (P.R. 170) with the ions [M+H]+ (m/z 455), [M+Na]+ (m/z 477) and the fragment ion m/z 318. As a further advantage of the NIST software compound specific information such as structural formula, molecular mass, synonyms, colour and CAS-no. can be added to the mass spectra library, and therefore also be used as search criterions. In the case of unknown mass spectra, the software can also extend the search to commercial GC-MS mass libraries (e.g. NIST main library).\n\nStandard addition and high-resolution mass spectrometry were used for the verification of particularly interesting pigments tentatively identified by NIST. As a quality control standard addition also allows checking for possible discriminations caused by the sample matrix. Verification is shown for a violet coloured tattoo ink containing the prohibited C.I. 51319 (P.V. 23), which was not declared (Figure 3). After being tentatively identified in a first run, spiking the sample with C.I. 51319 (Figure 3B) showed the same typical molecular ion cluster [M]+ (m/z 588) and two fragment ion clusters with m/z 554 and m/z 520 as in the original sample (Figure 3A). Instead of C.I. 51319, the product label listed two legal pigments red C.I.12477 (P.R. 210) and blue C.I.74160 (P.B. 15) both non-present in the ink. MALDI spectra did not reveal their characteristic ions [M+Na]+ (m/z 463) and [M+Na]+ (m/z 477) for the red pigment (a mixture of C.I. 12474 and 12475) and the ion cluster [M+H]+ (m/z 575) of the blue one (Figure 3C). This demonstrates a typical case of label forging for tattoo inks.\n\nVerification of the prohibited violet pigment C.I. 51319 in a tattoo ink sample by high resolution mass spectra (A) and standard addition (B), as well as the exclusion of a legal red (C.I. 12477) and blue (C.I. 74160) reference pigment (C) in the sample.\n\nIn the case of some 3,3’-dichlorobenzidine based diazodiaryl-pigments, only weak molecular ion mass signals ([M]+, [M+H]+, [M+Na]+) but more pronounced fragmentation were observed. In order to enhance molecular mass signals, three of the most often used MALDI- matrices, dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CCA), and sinapic acid (SA), were added to ink samples containing dichlorobenzidine based pigments. It turned out that all three had a positive effect, with DHB showing the best signal enhancement. The effect of DHB on the mass spectra of yellow pigment C.I. 21090 is shown in Figure 4. With DHB the signal intensity of the molecular ion [M]+ (m/z 628) was twice that of the sodium adduct [M+Na]+ (m/z 651), even twelve times as high than without matrix supplement.\n\nLDI mass spectra of pigment Yellow 12 (C.I. 21090) without matrix (A) and MALDI spectra of the same pigment with DHB as a matrix (B).\n\nBecause the identification of pigments by LDI/MALDI-TOF MS is a qualitative method, validation was only performed for mass resolution, accuracy of pigment identification, a rough estimation of limits of detection and the precision of characteristic mass intensity.\n\nAt the mass of 450 amu, a mass resolution of R = M/ΔM = 6000 (full width at half maximum, FWHM) was achieved. The resolution quality depends on the laser power and is not the same for all pigments. Generally, mass resolution decreases with higher laser power and a shift to a higher mass of up to 0.1 u can be observed.\n\nAccuracy of pigment identification in the screening mode (NIST, without sample washing and spiking) was tested with 48 different tattoo ink samples, their composition being unknown to the analyst. The results were checked for conformity using trustworthy declarations and/or additional in-house HPLC analysis of more or less soluble colourants. In 44 of the 48 samples (92%) all organic pigments were identified correctly (up to four in the same sample). Incomplete detection was found in two inks where the banned orange pigment C.I. 12075 remained undetected due to its weak signals in the mass spectra. The resulting poor library search matches did not give sufficient evidence for a positive identification. With the characteristic ion [M+H]+ (m/z 339), this pigment is usually well detectable. Therefore, a pigment concentration near the limit of detection is the most likely explanation for the weak signal. For the two other samples, results of LDI-TOF MS were not consistent with those of HPLC-analysis: in the first case, C.I. 21110 was identified with LDI-TOF MS, whereas HPLC-analysis gave C.I. 21115. In the second case C.I. 21100 was mistaken for C.I. 21095 because the characteristic mass signals of C.I. 21100 (m/z 522 and 550) were quenched.\n\nThe limits of detection (LOD) in a blue tattoo ink were estimated by standard addition with two red pigments, of which one was a well (C.I. 56110) and the other a poorly ionisable one (C.I. 14720). A correct identification of the pigment specific mass clusters by the NIST library was possible between sample concentrations of 1% (w/w) for the well ionisable pigment and of 10 – 20% (w/w) for the other one (C.I. 14720). As pigments are added to tattoo inks in the percent range, screening with LDI-TOF MS proved to have a suitable LOD.\n\nNevertheless, we have to consider that sometimes the LOD can also be affected by discrimination effects of tensides or inorganic pigments, such as carbon black (C.I. 77266) or TiO2 (C.I. 77891, white), present in the sample16. Whereas tensides can be easily removed by EtOH the separation of the two inorganic pigments remain an unresolved problem up to this date. However, with the application of a higher laser energy and colour plausibility checks, negative identification results due to matrix effects could be eliminated for all forbidden organic pigments.\n\nPrecision was determined as repeatability with ten determinations of the characteristic mass intensity of two pigments (C.I. 21108, C.I. 74260) and two different inks containing C.I.74160 and C.I.12475, respectively. The pigment specific relative standard deviations were in a range of 4% to 13%, which are by far sufficient for identification purposes. This good reproducibility is in contrast to common experience with MALDI-TOF-MS as inhomogeneous crystallisation of the matrix promotes the building of so called “hot-spots” with locally higher ionisation signals17. Obviously, the dispersion of the pigments spotted on the target plate using MeOH instead of a matrix seems to give a more homogenous distribution of the analytes. Good reproducibility was probably also due to laser scanning in the serpentine style over the whole sample with 20 accumulated laser shots per profile which evens out some inhomogeneity.\n\nOver the last eight years we used LDI/MALDI-TOF MS for market survey purposes in Switzerland. Samples were randomly but also risk based collected from tattooing and PMU studios and from different importers. Table 1 gives an overview of the organic pigments identified in tattoo ink and PMU samples. Data obtained show that pigment Blue 15 was the most often found colourant followed by the prohibited Pigment Green 7. All prohibited pigments, especially PG7, were more frequently found in inks (3 – 13%) than in PMUs (2 – 4%). In contrast to detected legal pigments where only 7% were missing on product labels of the samples, the prohibited pigments were often not declared (68%), for details see 18. This clearly gives evidence that label forging is widespread. This fraud considers pigments that are banned due to toxicological concerns and implications for consumer health may be serious, showcasing the urgency for widespread market controls.\n\nProhibited colours are marked with an .\n\n\nConclusion\n\nTo our knowledge, no study reported a LDI/MALDI-TOF MS method for the identification of colour pigments in tattoo inks, PMUs or cosmetic products. Method validation and using the MALDI method for market surveys demonstrated that the method described is fit for purpose. Therefore, LDI/MALDI-TOF MS is a further powerful tool particularly in combination with py-GC/MS10, HPLC or ultraviolet–visible spectroscopy18 for enforcing legal restrictions on pigments in tattoo inks and PMUs. It should be kept in mind, however, that the presented method gives no information on the levels of the detected organic pigments. For this, further studies would be necessary.\n\n\nData availability\n\nAs a law enforcement body, we are bound to official secrecy. Scrutinising and anonymising data was only done within an acceptable effort. Considering these restrictions, we provide raw data for all experiments presented in our work (Figure 1–Figure 4) and of the 18 most frequent pigments of the market surveys, as shown in Table 1. Further, we created an Excel sheet (Dataset 4) with intermediary data of the market surveys.\n\nF1000Research: Dataset 1. MALDI raw data are provided as MASTER_RUN-, CAL-, COR-, LBL-, RAW, RUN, STATS- and UAP- files using Launchpad™ version 2.9.3 software (Shimadzu-Biotech Corp., Kyoto). The data include the raw high resolution spectra of Figure 1, Figure 3 and Figure 4, 10.5256/f1000research.13035.d18476619\n\nF1000Research: Dataset 2. MALDI raw data are provided as TXT-files in ASCII-format using Launchpad™ version 2.9.3 software (Shimadzu-Biotech Corp., Kyoto). The data include the high resolution raw spectra of Figure 1, Figure 3 and Figure 4, 10.5256/f1000research.13035.d18476720\n\nF1000Research: Dataset 3. NIST spectrum peak lists are provided as MSP-files in ASCII-format with a mass resolution of 1 D (msp) using the software of NIST 2.0 (USA). The data include the NIST spectra of Figure 2, Table 1 and method validation (HPLC verification), 10.5256/f1000research.13035.d18476821\n\nF1000Research: Dataset 4. Intermediary data of Table 1 and a list of suppliers of pigments of Table 1 are provided as Excel-files (XLS). The data include sample-no, declaration of prohibited and legal pigments and reversed match values of pigments by NIST-software (Rfit), 10.5256/f1000research.13035.d18476922\n\nF1000Research: Dataset 5. An example of the construction of our home-made spectrum library using the NIST software 2.0 (USA) is shown as JPG-file. The data include compound name, structural formula, molecular mass, CAS-no., analysis date, intensity of the main spectrum peaks and synonyms, 10.5256/f1000research.13035.d18477023", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank Bernard Roux (State Laboratory Basel-City) for analytical support and helpful suggestions.\n\n\nReferences\n\nPiccinini P, Contor L, Pakalin S, et al.: Safety of tattoos and permanent make-up. State of play and trends in tattoo practices. JRC technical report. EUR 27528 EN; 2015. Publisher Full Text\n\nCouncil of Europe: Resolution ResAP(2008)1 on requirements and criteria for the safety of tattoos and permanent make-up. (Superseding resolution ResAP(2003)2 on tattoos and permanent make-up). Council Resolution of 20 February 2008. Accessed 15 Sep 2017. Reference Source\n\nVerordnung des EDI über Gegenstände für den Schleimhaut-, Haut- und Haarkontakt sowie über Kerzen, Streichhölzer, Feuerzeuge und Scherzartikel (817.023.41). Bern, 2005. Accessed 15 Sep 2017. Reference Source\n\nLehner K, Santarelli F, Penning R, et al.: The decrease of pigment concentration in red tattooed skin years after tattooing. J Eur Acad Dermatol Venereol. 2011; 25(11): 1340–1345. PubMed Abstract \| Publisher Full Text\n\nSchreiver I, Hesse B, Seim C, et al.: Synchrotron-based ν-XRF mapping and μ-FTIR microscopy enable to look into the fate and effects of tattoo pigments in human skin. Sci Rep. 2017; 7(1): 11395. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHauri U, Hohl C: Photostability and breakdown products of pigments currently used in tattoo inks. Curr Probl Dermatol. 2015; 48: 164–169. PubMed Abstract \| Publisher Full Text\n\nPoon KWC, Dadour IR, McKinley AJ: In situ chemical analysis of modern organic tattooing inks and pigments by micro-Raman spectroscopy. J Raman Spectrosc. 2008; 39(9): 1227–1237. Publisher Full Text\n\nVila A, Garcia JF: Analysis of the chemical composition of red pigments and inks for the characterization and differentiation of contemporary prints. Analytical Letters. 2012; 45(10): 1274–1285. Publisher Full Text\n\nHutton Carlsen K, Køcks M, Sepehri M, et al.: Allergic reactions in red tattoos: Raman spectroscopy for 'fingerprint' detection of chemical risk spectra in tattooed skin and culprit tattoo inks. Skin Res Technol. 2016; 22(4): 460–469. PubMed Abstract \| Publisher Full Text\n\nSchreiver I, Hutzler C, Andree S, et al.: Identification and hazard prediction of tattoo pigments by means of pyrolysis-gas chromatography/mass spectrometry. Arch Toxicol. 2016; 90(7): 1639–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStachura S, Desiderio VJ, Allison J: Identification of organic pigments in automotive coatings using laser desorption mass spectrometry. J Forensic Sci. 2007; 52(3): 595–603. PubMed Abstract \| Publisher Full Text\n\nSoltzberg LJ, Hagar A, Kridaratikon S, et al.: MALDI-TOF mass spectrometric identification of dyes and pigments. J Am Soc Mass Spectrom. 2007; 18(11): 2001–2006. PubMed Abstract \| Publisher Full Text\n\nBoon JJ, Learner T: Analytical mass spectrometry of artists’ acrylic emulsion paints by direct temperature resolved mass spectrometry and laser desorption ionisation mass spectrometry. J Anal Appl Pyrolysis. 2002; 64(2): 327–344. Publisher Full Text\n\nWyplosz N: Laser desorption mass spectrometric studies of artists' organic pigments. PhD Thesis University of Amsterdam; 2003. Reference Source\n\nDirks M: Making Innovative Tattoo Ink Products with Improved Safety: Possible and Impossible Ingredients in Practical Usage. In: Serup J, Kluger N, Bäumler W, editors. Tattooed Skin and Health. Curr Probl Dermatol. Basel: Karger AG; 2015; 48: 118–127. Publisher Full Text\n\nLechner L: Analyse von Pigmenten in Tätowiertinten mit MALDI-TOF-MS. Wissenschaftliche Abschlussarbeit Universität Hohenheim Stuttgart; 2015.\n\nPersike M: Verfahrensentwicklung zur Analytik kleiner Moleküle mittels MALDI-Massenspektrometrie. Dissertation Johann Wolfgang Goethe-Universität Frankfurt am Main; 2010. Reference Source\n\nHauri U: Inks for tattoos and permanent make-up—pigments, preservatives, aromatic amines, polyaromatic hydrocarbons and nitrosamines. Swiss National Investigation Campaign 2014. Department of Health, Kanton Basel-Stadt. 2014.\n\nNiederer M, Hauri U, Kroll L, et al.: Dataset 1 in: Identification of organic pigments in tattoo inks and permanent make-ups using MALDI-TOF mass spectrometry. F1000Research. 2017. Data Source\n\nNiederer M, Hauri U, Kroll L, et al.: Dataset 2 in: Identification of organic pigments in tattoo inks and permanent make-ups using MALDI-TOF mass spectrometry. F1000Research. 2017. Data Source\n\nNiederer M, Hauri U, Kroll L, et al.: Dataset 3 in: Identification of organic pigments in tattoo inks and permanent make-ups using MALDI-TOF mass spectrometry. F1000Research. 2017. Data Source\n\nNiederer M, Hauri U, Kroll L, et al.: Dataset 4 in: Identification of organic pigments in tattoo inks and permanent make-ups using MALDI-TOF mass spectrometry. F1000Research. 2017. Data Source\n\nNiederer M, Hauri U, Kroll L, et al.: Dataset 5 in: Identification of organic pigments in tattoo inks and permanent make-ups using MALDI-TOF mass spectrometry. F1000Research. 2017. Data Source" }	[ { "id": "28230", "date": "08 Dec 2017", "name": "Guido Vogel", "expertise": [ "Reviewer Expertise Protein mass profiling using MALDI-TOF MS for the identification bacteria", "fungi and eucaryotic cell lines" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by Niederer et al. on the \"Identification of organic pigments in tattoo inks and permanent make-ups using MALDI TOF MS“ is very well written and discusses a new approach for the detection of potentially dangerous organic pigments in tattoo inks.\nThe new method applying LDI- and MALDI-TOF MS is a big improvement as compared to those applied so far and will be of great help to authorities in their responsibility to regulate the use these pigments in tattoo studios. The importance of the availability of such methods is underlined by the very strong increase of persons with tattoos or permanent make-up in the last years.\nThe applicability of this new identification method was clearly demonstrated in the many years of testing it with real life samples.\nI do not have any suggestions for improving the text and the figures. They are perfect in my opinion the way they are.\nOnly the title could be improved by stating the use of LDI-TOF MS next to MALDI-TOF MS.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3291", "date": "08 Jan 2018", "name": "Markus Niederer", "role": "Author Response", "response": "We would like to thank the reviewer for his time and constructive feedback. As requested the title and some text passages were changed to clarify that LDI- instead of MALDI-TOF-MS was used in most cases." } ] }, { "id": "28228", "date": "11 Dec 2017", "name": "Christoph Hutzler", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe method article from Niederer et al. describes the identification of organic pigments in the complexes matrices of tattoo ink and permanent make-up using Matrix assisted laser desorption/ionization (MALDI) mass spectrometry. The authors created a reference library of >150 colorants and analyzed a total of 451 inks. The qualitative method described in this article was validated and counter checked with a complementary HPLC analysis suitable for soluble colorants.\nThe method is highly valuable to identify tattoo pigments in the course of market surveys. Their results show, that false declaration and fraud are frequent among tattoo and permanent make-up inks.\nIn the following minor amendments are suggested:\nDespite the common use of the plural form in the abbreviation PMUs, make-up itself comes without a plural and is considered uncountable.\n\nThe authors used both the color index and names of pigments to facilitate readability for scientists from different fields. In the section market survey an additional abbreviation PG7 appears. This might be replaced by either of the other identifiers.\n\nIn the abstract and section \"market survey\" the authors refer to the ban of pigments because of the hazardous properties and toxicological concerns. Although this might be correct for some pigments, the ban for tattoo inks might also derive from missing toxicological data instead of data claiming hazards, e.g. in the case of pigment green 7.\nIn summary, the presented method is of major value not only in the field of tattoo and permanent make up compliance, but may also be applied to cosmetic products or used in forensic analyses.\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3292", "date": "08 Jan 2018", "name": "Markus Niederer", "role": "Author Response", "response": "We thank the reviewers for their constructive comments regarding our study. As requested, the plural of make-up was corrected. In the section “market survey” the abbreviation PG7 was replaced by Pigment Green 7. In the abstract and section \"market survey\" text passages regarding the ban of pigments were modified." } ] } ]	1	https://f1000research.com/articles/6-2034
https://f1000research.com/articles/6-1780/v1	28 Sep 17	{ "type": "Research Article", "title": "Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia", "authors": [ "Reno K Kamarlis", "Muhammad ND Lubis", "Bethy S Hernowo", "Harapan Harapan", "Azmi S Kar", "Muhammad ND Lubis", "Bethy S Hernowo", "Azmi S Kar" ], "abstract": "Background: Using immunohistochemical stains to target specific breast cancer markers has become indispensable for evaluation of small diagnostic tissue specimens, and therefore novel marker cocktails for specific breast cancers are required. This study was conducted to assess the immunoexpression of P63 and SOX2 in triple negative breast cancer (TNBC), and to evaluate the predictive diagnostic value of these markers for specific types of TNBC. Methods: Histological slides and paraffin blocks of TNBC cases were collected from Dr. Hasan Sadikin Hospital, Bandung, Indonesia from 5-years period (2011-2015). Each histological slide was subjected to immunohistochemical staining for P63 (nucleus and cytoplasm) and SOX2 (nucleus), with specific primer antibodies. Immunoexpression of P63 and SOX2 was evaluated using immunoreactivity scoring. Associations between P63 and SOX2 immunoexpression and TNBC types were assessed using Mann Whitney tests. In addition, the predictive diagnostic values of these markers were assessed. Results: Forty TNBC histological slides were included, and 23 (57.5%) were Basal-like type TNBC and 17 (42.5%) were Non basal-like type TNBC. Immunoexpression of P63 nucleus and SOX2 was not different between types of TNBC. However, immunoexpression of P63 in the cytoplasm in Basal-like type TNBC was significantly higher than in Non basal-like type TNBC (p=0.021). Predictor diagnostic value analysis suggested that immunoexpression of P63 in cytoplasm had 56.5% sensitivity and 70.6% specificity for diagnosing Basal-like type TNBC, with area under curve of 0.64.\n\nConclusions: Immunoexpression of P63 in the cytoplasm has a relatively weak diagnostic value to discriminate Basal-like and Non basal-like types of TNBC.", "keywords": [ "breast cancer", "breast cancer marker", "P63", "SOX2", "triple negative breast cancers" ], "content": "Introduction\n\nBreast cancer, accounting for 25% of all cancer cases and 15% of all cancer deaths among females, is the most frequently diagnosed cancer among female worldwide1,2. In 2012, it was estimated that there were 1.7 million breast cancer cases with more than half million deaths. Between 1980s and 1990s, the incidence of breast cancer increased significantly, approximately by 30% in developed countries3 and currently it has been also rising in many developing countries2. In Asia, 639,824 breast cancer cases and 228,926 deaths were recorded in 2012, from which 48,998 cases and 19,750 deaths occurred in Indonesia4.\n\nAdvanced screening and diagnosis methods for breast cancer such as mammograms, ultrasound, magnetic resonance imaging and fine-needle aspiration, have allowed for detection of small lesions at the early stage. Identifying breast cancer at the early stage will increase the potential for curative treatment and therefore increases the survival rate5–8. However, smaller lesions are more challenging to diagnose. Therefore, it is essential to use an advanced immunohistochemical approach for evaluation of smaller tissue specimens that target more specific markers9.\n\nA previous study using a MCF7 breast cancer cell line to produce MCF7-derived tumour xenografts found that P63 and SOX2 immunostainings were two potential markers for breast cancer10. P63, involved in cellular differentiation, is a homolog of tumour protein P53 and in normal breast ducts and lobules it is expressed frequently in the nuclei of myoepithelial cells11. Mutation of the p53 gene results in a very high risk of breast cancer12. A study revealed that the total percentage of P63-positive cells was related to marked nuclear pleomorphism and the intensity of P63 staining was associated with syncytial growth pattern in triple-negative breast cancer (TNBC)13. In addition, data also reveals that p63 gene expression in breast cancer could be used as a specific marker of metaplastic carcinoma11, and P63 immunohistochemical staining could improve diagnostic accuracy of breast cancer even in small tissue specimens14.\n\nSOX2 is a transcription factor belonging to the SOX family and functions as an activator or suppressor of gene transcription15,16. Data shows that SOX2 promotes cellular proliferation of breast tissue17 and regulates self-renewal in cancer stem cells18. The scientific evidence reveals that SOX2 acts as an oncogene in epithelial cancers16 and in the breast, a study found that silencing of sox2 gene was associated with reduction of the size of the cancer stem cells and restoration of tamoxifen sensitivity19. All together, these data indicate that P63 and SOX2 have pivotal role in breast cancer and therefore are potential to be used as specific biomarkers. This study was conducted to assess the immunoexpression of P63 and SOX2 in TNBC cases in order to provide insight regarding their potential diagnostic value (single or in combination) to differentiate TNBC types.\n\n\nMethods\n\nA cross-sectional study to assess the immunoexpression of P63 and SOX2 in TNBC cases (negative expression of estrogen and progesterone receptors and c-erbB2) was conducted. Histological slides of TNBC and their paraffin blocks, tested between the 1st of January 2011 and 31st of December 2015, were collected from the Pathology Anatomy Laboratory, Dr. Hasan Sadikin Hospital, Bandung, Indonesia. Each histological slide was examined by two certified pathologists. To classify the type of TNBC morphology, between Basal-like type TNBC and Non basal-like type TNBC, cytokeratin 5/6 (CK 5/6) immunohistochemical staining was carried out on all TNBC histological slides. Concurrently, the immunoexpression of P63 and SOX2 was measured using immunohistochemical stains with specific primer antibodies. The protocol of this study was approved by the Health Research Ethical committee of Sumatera Utara University (approval 103/KOMET/FK USU/2015) and the usage of histological specimens was approved by the Pathology Anatomy Laboratory of Dr. Hasan Sadikin Hospital (LB.02.01/B29/239/X/2015).\n\nForty archival paraffin blocks from TNBC cases were subjected to immunohistochemical staining to assess the immunoexpression of CK 5/6, P63 and SOX2. Briefly, 4 μm sections of each paraffin block were prepared using standard procedure20. Immunohistochemical staining was conducted using primary antibodies as follows: anti-CK5/6 monoclonal antibody (Biocare Medical, Concord, CA, USA), anti-P63 monoclonal antibody (Biocare Medical, Concord, CA, USA) and anti-SOX2 monoclonal antibody (Abcam, Cambridge, UK). Starr Trek Universal HRP Detection (Biocare Medical, Concord, CA, USA) was used as second antibody. A chromogen 3,3’-diaminobenzidine (DAB) (Biocare Medical, Concord, CA, USA) was used to develop the colour. For each experiment, appropriate controls were used.\n\nImmunoexpression of CK 5/6 was interpreted as positive or negative, in which positive CK 5/6 indicates Basal-like type TNBC while negative CK 5/6 indicates Non basal-like type TNBC. Immunoexpression of P63 and SOX2 was evaluated using an immunoreactivity scoring system that had been published elsewhere with modification13. Staining intensity was scored as follows: 1 (no staining), 2 (weak staining), 3 (moderate staining) and 4 (strong staining). The percentage of positively stained tumour cells was assessed as a proportion of the total number of tumour cells present in the section as follows: 1 (<20%), 2 (≥20–50%), 3 (>50–80%) and 4 (>80%).\n\nImmunoreactivity score was calculated by multiplying staining intensity and the percentage of positivity, and the score therefore ranged from 1 to 16. The immunoreactive score was then divided into low (≤ 5), moderate (≥ 6 – 10) and high (≥11 – 16). Immunoexpression of P63 was measured both in cytoplasm and nucleus while SOX2 immunoexpression was measured in nucleus only.\n\nNormality of the data was assessed using the Shapiro-Wilk test and therefore the analysis tests chosen based on the normality of the data. The correlations between immunoexpression of P63 (cytoplasm and nucleus) and SOX2 were assessed using Pearson correlation and Spearman correlation, respectively. The associations of P63 and SOX2 immunoexpression and type of TNBC were assessed using Mann Whitney test. The predictive diagnostic values of P63 cytoplasm for diagnosing Basal-like type TNBC were estimated using several immunoreactivity score cut-off points. Receiver operating characteristic curve (ROC) was plotted and area under the ROC curves (AUC) was estimated. For all analyses, estimates were considered statistically significant for two-tailed values of p<0.05. All analyses were conducted using Statistical Package for the Social Sciences software (SPSS for Windows, Version 16, Chicago, IL).\n\n\nResults\n\nThe histopathology of the TNBC samples used in this study is described in Table 1. Approximately 45% of the samples were classified as metaplastic carcinomas. In addition, immunohistochemical staining for CK 5/6 revealed that 23 (57.5%) of samples were Basal-like type TNBC and while 17 (42.5%) samples were Non basal-like type TNBC.\n\nImmunoexpression of P63 and SOX2 in samples, categorized by immunoreactivity score, are presented in Table 2. For both types of TNBC (basal and non basal-like type), all immunoreactivity scores for P63 in the nucleus were classified as low grade, while 11 (27.5%) and 7 (17.5%) samples were classified as moderate and high grade, respectively for the P63 in the cytoplasm. The immunoreactivity grade for SOX2 was similar to P63 in the cytoplasm, and therefore correlation analyses were conducted.\n\nThere was a strong negative correlation between immunoexpression of P63 in the cytoplasm and immunoexpression of SOX2 in the nucleus in metaplastic carcinoma (a sub-type of TNBC basal-like type) (r=-0.73, p=0.013) (Table 3). In addition, linear regression showed a relatively strong correlation between P63 cytoplasm and SOX2 immunoexpression in metaplastic carcinoma (r=0.49, p=0.012). There was no significant correlation between P63 cytoplasm and SOX2 immunoexpression in Non basal-like type TNBC, and no significant correlation between P63 nucleus and SOX2 immunoexpression either in Basal-like type or Non basal-like type of TNBC.\n\nSignificant at 0.05\n\nImmunoexpression of P63 cytoplasm, P63 nucleus and SOX2 in Basal-like and Non Basal-like TNBC is shown in Table 4. The data indicates that the immunoexpression of P63 cytoplasm in Basal-like type TNBC was significantly higher compared to Non basal-like type TNBC (p=0.021). Immunoexpression of P63 nucleus and SOX2 was not different between Basal-like and Non basal-like types of TNBC, with p-values of p=0.27 and p=0.17, respectively.\n\nSignificant at 0.05\n\nAs mentioned above, immunoexpression of P63 in the cytoplasm was the only marker that was significantly different between TNBC types. Therefore, immunoreactivity score of P63 cytoplasm was further analysed to determine its ability to predict Basal-like type TNBC. Table 5 shows the predictive values of P63 in the cytoplasm for determining Basal-like type TNBC, using seven immunoreactivity score cut-off values from 3 to 9. It shows that P63 has relatively weak diagnostic value in diagnosing Basal-like type TNBC. The highest sensitivity was achieved at immunoreactivity score 3, while specificity was increasing with a higher immunoreactivity score.\n\nUsing the average score of P63 cytoplasm immunoexpression for Basal-like type TNBC in this study, 5.6 or 6, the sensitivity and specificity of P63 cytoplasm immunoreactivity score to predict Basal-like type TNBC was 56.5% and 72.6%, respectively with area under curve 0.64. The receiver operating curve of predictive diagnostic value of P63 cytoplasm for determining Basal-like type TNBC is plotted in Figure 1.\n\n\nDiscussion\n\nTo the best of our knowledge, this is the first study conducted to assess the immunoexpression of P63 and SOX2 in TNBC cases in Indonesia. Some studies have been conducted to assess the predictive values of P63 as specific marker for breast cancer11,21. In addition, the idea of utilization of a cocktail of specific markers has been proposed previously to provide higher sensitivity and specificity for diagnosing specific breast cancers9,13,22. However, none of the previous studies had been conducted to assess the diagnostic value of immunoexpression of P63 and SOX2 in combination. This study, at the beginning, sought to assess predictive value of combination both of those markers for specific type of TNBC. However, we found that there was no difference in immunoexpression of SOX2 between Basal-like type TNBC and Non basal-like type TNBC. Nevertheless, we found that immunoexpression of P63 cytoplasm, but not P63 nucleus, was higher in Basal-like type TNBC compared to Non basal-like type TNBC.\n\nP63 is highly expressed in embryonic ectoderm and in the nuclei of basal regenerative cell of many epithelial tissues23. P63 is expressed selectively in basal mammary epithelial cells and its expression is increased during mammary gland maturation24. P63 has been proposed as a breast cancer marker for a long time, but with conflicting results. A study demonstrated that immunoexpression of P63 was associated with breast cancers, for example the metaplastic carcinoma type of breast cancer11, but there was no difference in immunoexpression of P63 between medullary breast carcinomas and atypical medullary breast carcinomas of TNBC21. In our study, we found that the sensitivity and specificity of P63 cytoplasm immunoexpression to diagnose Basal-like type TNBC was 56.5% and 72.6%, respectively, with area under curve of 0.64. This sensitivity and specificity seems higher compared to a previous study, with 14% and 94%, respectively in determining a Basal-like type in infiltrative ductal carcinomas (TNBC)13. All together, these data indicate a weak predictive value of P63 immunoexpression as marker for Basal-like type TNBC. However, a study found that P63 is a specific marker for metaplastic carcinomas of the breast (a sub-type of Basal-like type TNBC)11. In our study, we could not assess the predictive value of P63 cytoplasm immunoexpression for determining metaplastic carcinomas, due to the small sample size (see Table 3).\n\nWe found that SOX2 immunoexpression grade was classified as moderate and high grade in 55% of TNBC cases (Table 2,) and it has been indicated previously that SOX2 has strong roles in promoting breast cancers17–19,25. However, there was no different in immunoexpression between Basal-like type TNBC and Non basal-like type TNBC. This result indicates that SOX2 expression is not different amongst TNBC types. This finding was in line with a previous study that indicated that SOX2 was expressed across different breast cancer subtypes26. A study found that SOX2 antibody in the sera was is higher in patients with breast cancer compared to healthy women and therefore it could be used to discriminate between breast cancer patients and healthy controls27. In addition, a meta-analysis found that SOX2 expression was associated with tumor size, histological grade, the aggressiveness and lymph node metastasis in TNBC patients28. All together, these results indicate that there was a possibility SOX2 expression could be used for diagnosing breast cancers, but there was no difference in expression amongst breast cancer types, and therefore it could not be used as specific marker for differentiating TNBC types.\n\nThere are some limitations to this study. The sample size was relatively small, and therefore some analyses could not be conducted. In addition, the diagnostic specimens were collected from different procedures such as from biopsy, mastectomy or lumpectomy, and this might influence the immunoexpression of the markers.\n\n\nConclusions\n\nImmunoexpression of P63 cytoplasm is higher among Basal-like type TNBC compared to Non basal-like type TNBC. However, the predictive diagnostic value of P63 immunoexpression in the cytoplasm for Basal-like type TNBC is relatively low, with 56.5% sensitivity and 72.6% specificity.\n\n\nData availability\n\nDataset 1: Immunoexpression and immunoreactivity scores of P63, SOX2 and CK 5/6 in the forty specimens that were analysed. DOI, 10.5256/f1000research.12671.d17913129", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKey TJ, Verkasalo PK, Banks E: Epidemiology of breast cancer. Lancet Oncol. 2001; 2(3): 133–140. PubMed Abstract \| Publisher Full Text\n\nTorre LA, Bray F, Siegel RL, et al.: Global Cancer Statistics, 2012. CA Cancer J Clin. 2015; 65(2): 87–108. PubMed Abstract \| Publisher Full Text\n\nAlthuis MD, Dozier JM, Anderson WF, et al.: Global trends in breast cancer incidence and mortality 1973–1997. Int J Epidemiol. 2005; 34(2): 405–412. PubMed Abstract \| Publisher Full Text\n\nGhoncheh M, Momenimovahed Z, Salehiniya H: Epidemiology, Incidence and Mortality of Breast Cancer in Asia. Asian Pac J Cancer Prev. 2016; 17(S3): 47–52. PubMed Abstract \| Publisher Full Text\n\nAIHW: Breast cancer survival by size and nodal status in Australia. In: Registries NBCCAAoC, ed. Cancer series no. 39. Canberra: AIHW; 2007. Reference Source\n\nAllemani C, Minicozzi P, Berrino F, et al.: Predictions of survival up to 10 years after diagnosis for European women with breast cancer in 2000–2002. Int J Cancer. 2013; 132(10): 2404–2412. PubMed Abstract \| Publisher Full Text\n\nAllemani C, Weir HK, Carreira H, et al.: Global surveillance of cancer survival 1995–2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet. 2015; 385(9972): 977–1010. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNaroda S, Iqbala J, Miller AB: Why have breast cancer mortality rates declined? J Cancer Policy. 2015; 5: 8–17. Publisher Full Text\n\nReisenbichler ES, Ross JR, Hameed O: The clinical use of a P63/cytokeratin7/18/cytokeratin5/14 antibody cocktail in diagnostic breast pathology. Ann Diagn Pathol. 2014; 18(6): 313–318. PubMed Abstract \| Publisher Full Text\n\nLiu Y, Coates PJ, Nenutil R, et al.: Lack of correlation between markers of breast cancer initiating cells. Breast Cancer Research. 2010; 12(Suppl 1): P38. Publisher Full Text\n\nKoker MM, Kleer CG: p63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma. Am J Surg Pathol. 2004; 28(11): 1506–1512. PubMed Abstract \| Publisher Full Text\n\nAssi HA, Khoury KE, Dbouk H, et al.: Epidemiology and prognosis of breast cancer in young women. J Thorac Dis. 2013; 5 Suppl 1: S2–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThike AA, Cheok PY, Jara-Lazaro AR, et al.: Triple-negative breast cancer: clinicopathological characteristics and relationship with basal-like breast cancer. Mod Pathol. 2010; 23(1): 123–133. PubMed Abstract \| Publisher Full Text\n\nHarton AM, Wang HH, Schnitt SJ, et al.: p63 Immunocytochemistry improves accuracy of diagnosis with fine-needle aspiration of the breast. Am J Clin Pathol. 2007; 128(1): 80–85. PubMed Abstract \| Publisher Full Text\n\nWeina K, Utikal J: SOX2 and cancer: current research and its implications in the clinic. Clin Transl Med. 2014; 3: 19. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSarkar A, Hochedlinger K: The sox family of transcription factors: versatile regulators of stem and progenitor cell fate. Cell Stem Cell. 2013; 12(1): 15–30. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStolzenburg S, Rots MG, Beltran AS, et al.: Targeted silencing of the oncogenic transcription factor SOX2 in breast cancer. Nucleic Acids Res. 2012; 40(14): 6725–6740. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeis O, Eguiara A, Lopez-Arribillaga E, et al.: Sox2 expression in breast tumours and activation in breast cancer stem cells. Oncogene. 2012; 31(11): 1354–1365. PubMed Abstract \| Publisher Full Text\n\nPiva M, Domenici G, Iriondo O, et al.: Sox2 promotes tamoxifen resistance in breast cancer cells. EMBO Mol Med. 2014; 6(1): 66–79. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAnderson G, Bancroft J: Tissue processing and microtomy. In: Bancroft JG, M., ed. Theory and practiceal of histological techniques 5th Edition. Edinburgh Churchill Livingstone; 2002; 109–123.\n\nMatkovic B, Juretic A, Separovic V, et al.: Immunohistochemical analysis of ER, PR, HER-2, CK 5/6, p63 and EGFR antigen expression in medullary breast cancer. Tumori. 2008; 94(6): 838–844. PubMed Abstract\n\nTacha DE, Bloom K, Kyshtoobayava A, et al.: A double immunostaining technique with a cocktail CK5, CK14, p63, CK7 and CK18 distinguishes between hyperplasia of the usual type, atypical hyperplasia, microinvasive and basal phenotype breast cancers. Modern Pathology. 2009; 22: 388a.\n\nWestfall MD, Pietenpol JA: p63: Molecular complexity in development and cancer. Carcinogenesis. 2004; 25(6): 857–864. PubMed Abstract \| Publisher Full Text\n\nForster N, Saladi SV, van Bragt M, et al.: Basal cell signaling by p63 controls luminal progenitor function and lactation via NRG1. Dev Cell. 2014; 28(2): 147–160. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiu K, Xie F, Gao A, et al.: SOX2 regulates multiple malignant processes of breast cancer development through the SOX2/miR-181a-5p, miR-30e-5p/TUSC3 axis. Mol Cancer. 2017; 16(1): 62. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLengerke C, Fehm T, Kurth R, et al.: Expression of the embryonic stem cell marker SOX2 in early-stage breast carcinoma. BMC Cancer. 2011; 11: 42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSun Y, Zhang R, Wang MJ, et al.: SOX2 autoantibodies as noninvasive serum biomarker for breast carcinoma. Cancer Epidemiol Biomarkers Prev. 2012; 21(11): 2043–2047. PubMed Abstract \| Publisher Full Text\n\nZheng Y, Qin B, Li F, et al.: Clinicopathological significance of Sox2 expression in patients with breast cancer: a meta-analysis. Int J Clin Exp Med. 2015; 8(12): 22382–22392. PubMed Abstract \| Free Full Text\n\nHarapan H, Reno KK, Muhammad NDL, et al.: Dataset 1 in: Immunoexpression of P63 and SOX2 in triple-negative breast cancers, Indonesia. F1000Research. 2017. Data Source" }	[ { "id": "26921", "date": "12 Oct 2017", "name": "Irianiwati Widodo", "expertise": [ "Reviewer Expertise I am a pathologist with major interest in breast cancer" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIntroduction:\n\nDo not explain the epidemiology of breast cancer too long, but explain more detail about TNBC and its subtypes (Basal-like and non basal-like), such as differences in histological type, behaviour, prognosis, treatment etc. So, the role of p63 and SOX2 on TNBC becomes clearer.\n\nMethod:\nStatistical analysis predictive diagnostic value of cytoplasmic p63 expression of basal-like and non basal-like TNBC, as far as I know there should be also negative p63 expression. Therefore I suggest to ask a qualified statistician.\n\nDiscussion:\nExplain the importance of negative correlation between p63 expression and metaplastic carcinoma. Compare with previous studies.\n\nConclusion:\nMention also other important results of this study such as:\nThe frequency of non basal-like is higher than basal-like. The most common histological type of TNBC is metaplastic carcinoma. Negative correlation between p63 expression and metaplastic carcinoma SOX2 expression in TNBC, even there is no stattistically significant\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3206", "date": "08 Jan 2018", "name": "Harapan Harapan", "role": "Author Response", "response": "Thank you for comments and suggestion. We have deleted some sentences related to the general introduction of breast cancer and added specific information related to TNBC. We would like to confirm that predictive diagnostic analysis in this study was conducted using qualified statistician. There is no evidence to support the importance of the negative correlation between immunoexpression of P63 cytoplasm and immunoexpression SOX2 nucleus in metaplastic carcinoma, and therefore we are unable to discuss this finding in depth. In conclusion section, we have added some additional principal finding as suggested by the reviewer." } ] }, { "id": "26782", "date": "20 Oct 2017", "name": "Diah Rini Handjari", "expertise": [ "Reviewer Expertise Pathologist with major interest in colorectal cancer" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI. - In introduction there is only little information regarding of nuclear expression of p63.\n\n- It doesn't mention about cytoplasmic p63 expression.\n\nWhat is significance function of p63 in tumor progression or disease progression?\n\nIt written in result and analysis.\n\n- In introduction you haven't discussed yet about the incidence of triple negative breast cancer in the worldwide, Asia or Indonesia ?\n\nII. In the methods, to classify TNBC morphology into Basal like and non Basal like, based on only, antibody (CK 5/6) but you didn't perform EGFR staining. Which is one of the marker of TNBC.\n\n- Is there any reference about the scoring of intensity and the percentage of positivity of expression of p63?\n\n- Is there any reference about imunoreactive score?\nIII. In discussion, there is also no information about cytoplasmic p63 and its role in disease progression.\n\n- Researcher only stated that cytoplasmic p63 expression has predictive value to classify breast cancer into TNBC and there is no comparison of p63 expression sensitivity and specificity with other TNBC marker as like CK 5/6 and EGFR which has been routinely used.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3205", "date": "08 Jan 2018", "name": "Harapan Harapan", "role": "Author Response", "response": "Thank you for comments and suggestion. We have deleted some sentences related to the general introduction of breast cancer and added specific information related to TNBC. We would like to confirm that predictive diagnostic analysis in this study was conducted using qualified statistician. There is no evidence to support the importance of the negative correlation between immunoexpression of P63 cytoplasm and immunoexpression SOX2 nucleus in metaplastic carcinoma, and therefore we are unable to discuss this finding in depth. In conclusion section, we have added some additional principal finding as suggested by the reviewer." }, { "c_id": "3207", "date": "08 Jan 2018", "name": "Harapan Harapan", "role": "Author Response", "response": "Thank you for comments and suggestion. We have deleted some sentences related to the general introduction of breast cancer and added specific information related to TNBC. In the revised manuscript, the data of the incidence from global, Southeast Asian countries and Indonesia are included.In our study, we used the histological paraffin blocks that have been confirmed as TNBC previously by testing ER, PR and HER2. To differentiate Basal like and non Basal like, either CK 5/6 or EGFR staining could be used with no significant different sensitivity and specificity (Livasy et al., 2006; Nielsen et al., 2004). In this study, CK 5/6 was employed to differentiate TNB into Basal like and non Basal like and this procedure was conducted during the study. This method is established method to define basal phenotype (Sasa et al., 2008; Rakha et al., 2007). In our study, we used the histological paraffin blocks that have been confirmed as TNBC previously by testing ER, PR and HER2. To differentiate Basal like and non Basal like, either CK 5/6 or EGFR staining could be used with no significant different sensitivity and specificity (Livasy et al., 2006; Nielsen et al., 2004). In this study, CK 5/6 was employed to differentiate TNB into Basal like and non Basal like and this procedure was conducted during the study. This method is established method to define basal phenotype (Sasa et al., 2008; Rakha et al., 2007). CK 5/6 and EGFR.As mentioned Immunohistochemistry Section, the principle of the scoring system for immunoexpression of P63 and SOX2 used in this study was have been elsewhere with modification (Thike et al., 2010). In detailed: Staining intensity was scored as follows: 1 (no staining), 2 (weak staining), 3 (moderate staining) and 4 (strong staining). The percentage of positively stained tumour cells was assessed as a proportion of the total number of tumour cells present in the section as follows: 1 (<20%), 2 (≥20–50%), 3 (>50–80%) and 4 (>80%). Then from staining intensity and percentage of positively stained cells, we created immunoreactivity score by multiplying staining intensity and the percentage of positivity. The score, therefore, ranged from 1 to 16 (divided into low (≤ 5), moderate (≥ 6 – 10) and high (≥11 – 16). However, specifications for staining intensity and the percentage of positivity used in this study are the standard system used by Pathology Anatomy Laboratory of Dr. Hasan Sadikin Hospital Bandung since 1990. These score systems are also adopted in some breast cancer diagnostic centres in Indonesia.Immunoreactive score system used in this study have been published elsewhere (Thike et al., 2010). We mentioned this in Immunohistochemistry Section of our Methods.In this study produce all raw data related to: type of TNBC (Basal like and Non Basal-like), subtype of cancer, expression of SOX2, P63 and CK 5/6 including staining intensity, distribution of the positively cells and immunoreactive score. Reference:Thike AA, Cheok PY, Jara-Lazaro AR, et al. Triple-negative breast cancer: clinicopathological characteristics and relationship with basal-like breast cancer. Mod Pathol. 2010;23(1):123–133.Livasy CA, Karaca G, Nanda R, et al. Phenotypic evaluation of the basal-like subtype of invasive breast carcinoma. Mod Pathol 2006;19:264-271Nielsen TO, Hsu FD, Jensen K, et al. Immunohistochemical and clinical characterization of the basal-like subtype of invasive breast carcinoma Clin Cancer Res, 2004;10:5367-5374Rakha EA, El-Sayed ME, Green AR, et al. Breast carcinoma with basal differentiation: a proposal for pathology definition based on basal cytokeratin expression. Histopathology. 2007 Mar;50(4):434-8.Sasa M, Bando Y, Takahashi M, et al. Screening for basal marker expression is necessary for decision of therapeutic strategy for triple-negative breast cancer. J Surg Oncol, 2008; 97: 30-34" } ] } ]	1	https://f1000research.com/articles/6-1780
https://f1000research.com/articles/7-22/v1	08 Jan 18	{ "type": "Software Tool Article", "title": "archiDART v3.0: A new data analysis pipeline allowing the topological analysis of plant root systems", "authors": [ "Benjamin M. Delory", "Mao Li", "Christopher N. Topp", "Guillaume Lobet", "Mao Li", "Christopher N. Topp", "Guillaume Lobet" ], "abstract": "Quantifying plant morphology is a very challenging task that requires methods able to capture the geometry and topology of plant organs at various spatial scales. Recently, the use of persistent homology as a mathematical framework to quantify plant morphology has been successfully demonstrated for leaves, shoots, and root systems. In this paper, we present a new data analysis pipeline implemented in the R package archiDART to analyse root system architectures using persistent homology. In addition, we also show that both geometric and topological descriptors are necessary to accurately compare root systems and assess their natural complexity.", "keywords": [ "archiDART", "plant root systems", "topology", "persistent homology", "Fitter indices", "Data Analysis of Root Tracings (DART)", "Root System Markup Language (RSML)" ], "content": "Introduction\n\nThe quantification of plant root systems is central in many research areas, ranging from developmental studies1 to crop phenotyping2. Root systems are often characterised using geometric descriptors, such as the total root length3, total root surface4, or number of root tips5. However, such descriptors often fail to describe the full complexity of root systems. Additional descriptors able to describe the topology of root systems are often missing, despite the fact that they can help understanding the feedback between plant morphology and root system functions6,7.\n\nThe topology of a root system is an important component of its architecture and refers to how individual roots are connected to each other through branching8. Studying the topology of branching structures as complex as root systems is challenging and requires quantitative methods allowing the description and comparison of plant morphologies7. In the 1980’s, A.H. Fitter introduced a method to describe branching structures and classify root systems into topologically distinct networks9. His method relies on the calculation of three indices to describe the topology of a root system, namely the magnitude, altitude, and external path length. A detailed description of this method can be found in 9,10. More recently, the use of persistent homology to quantify plant morphologies was introduced in the plant sciences community. This method was successfully used to quantify leaf shapes, leaf serrations, and root system architectures11. Persistent homology is a mathematical framework allowing the quantification of plant morphologies at different scales (from organs to organisms). Because plant roots can be represented as a succession of nodes connected by straight lines in a tree graph, they are referred to as zero-order homology groups (H0, path-connected component) in mathematics. The goal of a persistent homology analysis applied to a root system is to study how H0 features persist across the scales of a continuous mathematical function. A common mathematical function used to capture the topology of branching structures, such as plant shoots and root systems, is the geodesic distance (i.e., the distance measured along the roots between the root system base and any point of the root system). A nice explanation of how persistent homology can be applied to capture plant topologies is provided in 12. The main output of a persistent homology analysis is a persistence barcode recording the birth (apparition of a new connected component) and death (fusion of two connected components) of each H0 branch when a distance function traverses the branching structure (Figure 1). The degree of similarity between different root system topologies can be assessed by computing a pairwise distance matrix using a bottleneck distance method to compare persistence barcodes. Multivariate statistical tools, such as multidimensional scaling, can then be used to visualize topological differences between root systems12.\n\nWhen two H0 branches merge, the longest one persists and the shortest one dies.\n\nIn the past decade, many tools were developed to analyse root systems from digital images (for an extensive list, see the plant-image-analysis.org website13) or model root system architectures14,15. Several of these tools are able to extract the full root system architecture from the images, including the topology16–19. A common format for the storage of root architecture data, the Root System Markup Language (RSML)20, was also created to facilitate the exchange of information between researchers. Building on this new format, several tools were created to analyse root architecture data20,21. Among these tools, the R package archiDART offers a wide range of functionalities to analyse root system architectures in a free, open-source, and popular data analysis environment21.\n\nIn this paper, we present a new version (v.3.0) of the R package archiDART. In comparison with the version described earlier21, this version now includes several topological analysis methods, including, but not limited to, persistent homology. Our main objective is to demonstrate how the functions of the archiDART package can be used to analyse and compare the topology of plant root systems using persistent homology. In addition, we also aim to show that the topological analysis of plant root systems is highly complementary to the more classical approach that uses a set of geometric descriptors to compare root systems.\n\n\nMethods\n\narchiDART is an R package developed for the automated analysis of plant root system architectures using Data Analysis of Root Tracings (DART)17 and Root System Markup Language files (RSML)20. The version 3.0 of archiDART can be downloaded from the CRAN repository. An overview of the functions available in the package is presented in Table 1. Among the 10 functions developed for the package, 5 were already presented elsewhere21 and will not be further discussed in this paper.\n\nThe italicized functions were already presented in 21. DART, Data Analysis of Root Tracings; RSML, Root System Markup Language.\n\nIn comparison with the version presented earlier, the version 3.0 of the package supports the analysis of 3D root systems. In addition, time series data in RSML files can be analysed if the root system age is stored as a continuous function along the root segments. Finally, we developed a set of 5 new functions and updated the architect function to allow the topological analysis of plant root systems. The architect function is now able to calculate the topological indices introduced by Fitter10, and the 5 new functions presented in this paper are devoted to the topological analysis of root systems using persistent homology12.\n\nAll functions of archiDART were coded using the R programming language. The package is compatible with Windows, Mac OS X, and major Linux operating systems. A detailed documentation file listing the package dependencies and describing all the functions listed in Table 1 can be downloaded from the CRAN package area. The bottleneckdist function of archiDART relies on the bottleneck function of the TDA package22 to compute the bottleneck distance between two persistence diagrams.\n\nThe root system library used in this paper has already been presented elsewhere23. Briefly, this library contains a total of 10,464 simulated root systems created using the root architecture model ArchiSimple14. The result of each simulation was stored as an RSML file. The library consists of two categories of root systems: tap-rooted (5212) and fibrous (5252). For the use cases presented in this paper, 50 tap-rooted and 50 fibrous root systems were selected from the RSML library. All root systems used in this paper had a total root length comprised between 17 and 23 m (20 m ± 15%). Summary statistics describing the root system library used in this study are presented in Table S1.\n\nIn order to demonstrate and illustrate the capabilities of archiDART, we developed a web application (archiShiny) using the Shiny library24. This application is freely available here: https://plantmodelling.shinyapps.io/archidart. We developed archiShiny with the following aims in mind: (1) demonstrating how multivariate statistical tools (such as principal component analysis) can be used on the aggregated metrics computed by the architect function to differentiate root systems; (2) showing how root systems can be plotted using the advanced graphical functions of the ggplot2 library25; and (3) comparing the topology of root systems using persistent homology. The web application uses a library of 70 RSML files created using the root architecture model ArchiSimple14. Based on the initial values of the parameters of the model, the root systems were classified into seven genotypes (mock, dense, sparse, steep, shallow, slow, and fast). Each genotype was represented by 10 simulations. The different genotypes were based on a standard parameter set (mock) and had one parameter changed: growth rate (slow vs. fast), inter-lateral distance (dense vs sparse) or gravitropism (steep vs shallow).\n\n\nUse cases\n\nAfter package installation, the topological analysis of plant root systems (RSML files) using persistent homology comprises four main steps: (1) creating an rsmlToTable object, (2) computing persistence barcodes, (3) computing a pairwise bottleneck distance matrix, and (4) visualizing topological differences between root systems using non-metric multidimensional scaling (NMDS). The main steps of the analysis performed in this section of the paper are summarized in Figure 2. Although we only present the analysis pipeline developed for RSML files, root systems vectorized with DART can be analysed using exactly the same approach (see Figure 2).\n\nThe archiDART functions are italicized and written in green. archiDART objects are written in orange. DART, Data Analysis of Root Tracings; RSML, Root System Markup Language; NMDS, non-metric multidimensional scaling.\n\nThe first step of the analysis is to import the RSML files into R with the rsmlToTable function. If root systems were vectorized with DART, the dartToTable function should be used instead. The rsmlToTable function creates a data frame (table) containing at least 23 columns (spatial coordinates, length, diameter, surface, volume, growth rate, orientation, geodesic distance, etc.) and as many lines as root segments. Here, a root segment is defined as the straight line between two nodes in the data file. The table is an rsmlToTable object that can directly be used as an input to compute the persistence barcodes using the perhomology function. It is worth noting that rsmlToTable objects can also be used as an input for the architect function of this new version of the package to compute a set of aggregated metrics describing the global architecture of plant root systems.\n\nThe perhomology function computes the persistence barcode of each root system stored in an rsmlToTable or a dartToTable object. Each persistence barcode is computed using a geodesic distance function (Figure 3). For each root system, the results are stored as a barcode object in a list that contains as many elements as root systems. A barcode object is a matrix with 3 columns (dimension, birth, and death) and has as many lines as zero-order homology bars in the persistence barcode. An S3 method (plot.barcode) was developed for plotting persistence barcodes. A code example to compute and plot persistence barcodes from RSML files is provided below.\n\nVertical lines indicate the position along the geodesic distance function (from left to right). The version of this figure in the online article is interactive and was produced with the plotly library26.\n\n\n\nTo compare persistence barcodes against each other, a pairwise distance matrix is needed and the bottleneck distance is one possible option. The bottleneck distance is considered as a robust dissimilarity metric between two persistence barcodes, and its interpretation is quite straightforward: the greater the distance between two persistence barcodes, the greater will be the dissimilarity between them12. Such pairwise bottleneck distance matrix can be calculated with the bottleneckdist function of the package. This function only requires a perhomology object as an input. It has to be noted that the computation time required to compute a bottleneck distance matrix is highly dependent on the number and complexity of root systems being compared. A code example to compute a bottleneck distance matrix from persistence barcodes is provided below.\n\n\n\nA large variety of morphological, architectural, and topological traits can be measured on plant root systems (e.g., total root length, diameter, number of lateral roots per branching order, lateral root density, Fitter indices, etc.). When working with root architecture models and image analysis tools supporting the RSML format, such traits can be easily extracted from RSML files using the architect function of the archiDART package21 or the ImageJ plugin RSML Reader20. Using multivariate statistical tools, such as principal component analysis (PCA), one can then determine the key traits differentiating the root systems being compared23. In the next section, we would like to show that the information gained with this approach can be nicely complemented by a topological analysis of root systems using persistent homology.\n\nAfter selecting 100 root systems from a large RSML library, we first used the architect function to compute a set of 20 traits for each root system (Table S1). Then, we performed a PCA to visualize differences between root systems and find the most interesting morphological, architectural, and topological variables to differentiate them. On the score plot constructed with the two first principal components, a good separation between fibrous and taproot root systems can be observed on the first principal component (Figure 4A). On average, fibrous root systems were characterized by a greater number/length of first-order roots, while taproot systems had a greater lateral root length and a greater secondary root density (Figure 4B, Table S2). Interestingly, two topological indices (altitude and external path length) were on average greater for taproot systems. On the second principal component, however, a separation between dicotyledonous root systems can be observed (Figure 4A). Negative PC2 scores were mainly associated with taproot systems having a greater number/length of tertiary roots and a greater magnitude, while root systems with positive PC2 scores had on average greater root diameters, surface, and volume (Figure 4B, Table S2). Although this approach is very useful to assess root system diversity and derive a functional classification of root systems27, it poorly takes into account topological differences that might exist between root systems sharing similar trait values.\n\nIn total, 100 root systems were considered for the analysis (50 fibrous and 50 tap-rooted). In the first approach, root systems were compared using a set of 20 traits computed by the architect function of archiDART. A PCA was then performed to visualize differences between root systems and find the most interesting traits to differentiate them (panels A and B). The PCA was performed on a correlation matrix constructed from scaled variables using the PCA function of the FactoMineR package28. In the second approach, we used persistent homology to compare the topology of root systems. Topological differences between root systems were visualized using non-metric multidimensional scaling (NMDS, panel C). The NMDS was performed on a pairwise bottleneck distance matrix with the metaMDS function of the vegan library29. In panel D, two persistence barcodes are compared. In panels A and C, each dot is a branching structure and four root system of interests are spotted using orange (taproot) and green (fibrous) crosses. Abbreviations used in panel B: TRL, total root length; L1R, total first-order root length; TN1R, number of first-order roots; TNLR, total number of lateral roots; TLRL, total lateral root length; N2LR, number of second-order roots; N3LR, number of third-order roots; L2LR, total second-order root length; L3LR, total third-order root length; MD1, mean first-order root diameter; MDLR, mean lateral root diameter; D2LR, second-order root density; Convexhull, convex hull area; Stot, total root surface area; Vtot, root system volume; ExtPathLength, external path length.\n\nTo illustrate this, we plotted four representative root systems from the RSML library used in this study (Figure 5). Although the global architecture and topology of these root systems clearly differ, fibrous 1 and fibrous 2, as well as taproot 1 and taproot 2, were poorly separated by the PCA (Figure 4A). Therefore, we performed a topological analysis of the root systems in our library using the persistent homology analysis pipeline described previously. Non-metric multidimensional scaling (NMDS) was used to visualize dissimilarities between persistence barcodes (Figure 4C and D). Results showed that (1) fibrous and taproot root systems can be clearly separated using persistent homology, and (2) strong topological differences exist between fibrous 1 and fibrous 2, as well as between taproot 1 and taproot 2, despite the fact that these root systems were not separated by the PCA. Altogether, these results showed that persistent homology is highly complementary to the more traditional approach consisting at using a set of aggregated metrics to compare root systems.\n\nThe colour code refers to the geodesic distance (cm).\n\n\nConclusions\n\nIn this paper, we presented a new analysis pipeline implemented in the R package archiDART to perform topological analysis of plant root systems using root architectural data (DART or RSML files). Using root architecture models, we showed that persistent homology is an efficient tool to capture and compare the topology of a large diversity of root systems. In addition, our results showed that the use of both geometric and topological descriptors are necessary to capture the natural complexity of plant root systems. Because topology is independent of transformation and deformation, the analysis pipeline described in this paper is highly flexible and can be used on data describing the architecture of 3D (e.g., root architecture models) and 2D (e.g., excavated root systems) root systems. Altogether, we believe that this great flexibility in root architecture data, the ease of use of the functions developed for the analysis pipeline presented in this paper, as well as the open-source nature of archiDART, make topological analysis of root systems widely accessible to the scientific community.\n\n\nData and software availability\n\nThe data and R codes used for the use cases presented in this manuscript are available: https://doi.org/10.5281/zenodo.111783630\n\nThe latest stable version of archiDART is available on the CRAN repository: http://cran.r-project.org/package=archiDART\n\nSource code available from: https://github.com/archidart/archidart\n\nArchived source code as at time of publication: https://dx.doi.org/10.5281/zenodo.111786431\n\nSoftware license: GNU GPL v2.0\n\nMore information about archiDART can be found on this website: https://archidart.github.io/\n\nThe web application illustrating the capabilities of archiDART is accessible at: https://plantmodelling.shinyapps.io/archidart\n\nThe data and codes used to make the web application are available: https://github.com/archidart/archishiny\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.113343532\n\nLicense for web application: GNU GPL v3.0", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was funded by the Chair of Ecosystem Functioning and Services, Leuphana University, Lüneburg, Germany.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe would like to thank Magdalena Julkowska (KAUST), Pierre Delaplace (Liège University, Gembloux Agro-Bio Tech, Belgium), and Loïc Pagès (INRA, France) for their insightful comments on the web application.\n\n\nSupplementary material\n\nTable S1. Descriptive statistics of the root system library used in this study. The RSML library consisted of 50 fibrous and 50 taproot root systems created using the ArchiSimple model14.\n\nClick here to access the data.\n\nTable S2. Principal component analysis: Correlation between each root system variable and the two first principal components. Correlation coefficients written in bold contributed significantly to a principal component (PC). We considered that a variable contributed significantly to a PC if its contribution (in %) was greater than the contribution that would have been expected if all variables contributed equally to a PC (threshold value equal to 5%). The topological indices used in the PCA were calculated following9,10.\n\nClick here to access the data.\n\n\nReferences\n\nJulkowska MM, Hoefsloot HC, Mol S, et al.: Capturing Arabidopsis root architecture dynamics with ROOT-FIT reveals diversity in responses to salinity. Plant Physiol. 2014; 166(3): 1387–402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDas A, Schneider H, Burridge J, et al.: Digital imaging of root traits (DIRT): a high-throughput computing and collaboration platform for field-based root phenomics. Plant Methods. 2015; 11: 51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelaplace P, Delory BM, Baudson C, et al.: Influence of rhizobacterial volatiles on the root system architecture and the production and allocation of biomass in the model grass Brachypodium distachyon (L.) P. Beauv. BMC Plant Biol. 2015; 15: 195. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFang S, Clark RT, Zheng Y, et al.: Genotypic recognition and spatial responses by rice roots. Proc Natl Acad Sci U S A. 2013; 110(7): 2670–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPound MP, Atkinson JA, Townsend AJ, et al.: Deep machine learning provides state-of-the-art performance in image-based plant phenotyping. Gigascience. 2017; 6(10): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBucksch A, Atta-Boateng A, Azihou AF, et al.: Morphological Plant Modeling: Unleashing Geometric and Topological Potential within the Plant Sciences. Front Plant Sci. 2017; 8: 900. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBalduzzi M, Binder BM, Bucksch A, et al.: Reshaping Plant Biology: Qualitative and Quantitative Descriptors for Plant Morphology. Front Plant Sci. 2017; 8: 117. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLynch J: Root Architecture and Plant Productivity. Plant Physiol. 1995; 109(1): 7–13. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFitter AH: The topology and geometry of plant root systems: influence of watering rate on root system topology in Trifolium pratense. Ann Bot. 1986; 58(1): 91–101. Publisher Full Text\n\nFitter AH: An architectural approach to the comparative ecology of plant root systems. New Phytol. 1987; 106(s1): 61–77. Publisher Full Text\n\nLi M, Frank MH, Coneva V, et al.: Persistent homology: a tool to universally measure plant morphologies across organs and scales. bioRxiv. 2017. Publisher Full Text\n\nLi M, Duncan K, Topp CN, et al.: Persistent homology and the branching topologies of plants. Am J Bot. 2017; 104(3): 349–353. PubMed Abstract \| Publisher Full Text\n\nLobet G, Draye X, Périlleux C: An online database for plant image analysis software tools. Plant Methods. 2013; 9(1): 38. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPagès L, Bécel C, Boukcim H, et al.: Calibration and evaluation of ArchiSimple, a simple model of root system architecture. Ecol Modell. 2014; 290: 76–84. Publisher Full Text\n\nSchnepf A, Leitner D, Landl M, et al.: CRootBox: A structural-functional modelling framework for root systems. bioRxiv. 2017. Publisher Full Text\n\nPound MP, French AP, Atkinson JA, et al.: RootNav: navigating images of complex root architectures. Plant Physiol. 2013; 162(4): 1802–14. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLe Bot J, Serra V, Fabre J, et al.: DART: a software to analyse root system architecture and development from captured images. Plant Soil. 2010; 326(1–2): 261–73. Publisher Full Text\n\nLobet G, Pagès L, Draye X: A novel image-analysis toolbox enabling quantitative analysis of root system architecture. Plant Physiol. 2011; 157: 29–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeitner D, Felderer B, Vontobel P, et al.: Recovering root system traits using image analysis exemplified by two-dimensional neutron radiography images of lupine. Plant Physiol. 2014; 164: 24–35. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLobet G, Pound MP, Diener J, et al.: Root system markup language: toward a unified root architecture description language. Plant Physiol. 2015; 167: 617–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelory BM, Baudson C, Brostaux Y, et al.: archiDART: an R package for the automated computation of plant root architectural traits. Plant Soil. 2016; 398(1–2): 351–65. Publisher Full Text\n\nFasy BT, Kim J, Lecci F, et al.: TDA: Statistical Tools for Topological Data Analysis. R package version 1.5.1. 2017. Reference Source\n\nLobet G, Koevoets IT, Noll M, et al.: Using a Structural Root System Model to Evaluate and Improve the Accuracy of Root Image Analysis Pipelines. Front Plant Sci. 2017; 8: 447. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChang W, Cheng J, Allaire J, et al.: shiny: Web Application Framework for R. R package version 1.0.5. 2017. Reference Source\n\nWickham H: ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York; 2016. Publisher Full Text\n\nSievert C, Parmer C, Hocking T, et al.: plotly: Create Interactive Web Graphics via “plotly.js”. R package version 4.7.1. 2017. Reference Source\n\nBodner G, Leitner D, Nakhforoosh A, et al.: A statistical approach to root system classification. Front Plant Sci. 2013; 4: 292. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHusson F, Josse J, Le S, et al.: FactoMineR: Multivariate Exploratory Data Analysis and Data Mining. R package version 1.39. 2017. Reference Source\n\nOksanen J, Blanchet FG, Friendly M, et al.: vegan: Community Ecology Package. R package version 2.4-4. 2017. Reference Source\n\nDelory BM, Li M, Topp CN, et al.: Data and R codes used in Delory et al (2017) F1000Research (Version v1.1) [Data set]. Zenodo. 2017. Data Source\n\nDelory BM, Li M, Topp CN, et al.: archiDART 3.0 (Version v3.0). Zenodo. 2017. Data Source\n\nDelory BM, Li M, Topp CN, et al.: archiShiny: a web app to demonstrate the capabilities of archiDART 3.0 (Version 1.0). Zenodo. 2017. Data Source" }	[ { "id": "29709", "date": "26 Jan 2018", "name": "Magdalena M. Julkowska", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Delory et al., \"archiDART v3.0: A new data analysis pipeline allowing the topological analysis of plant root systems\" describes an R package archiDART that includes methods allowing analysis root topology using persistent homology. The paper describes the novelty of the package really well and it is providing new and exciting ways for the community to differentiate between various root architecture types. The Shiny app developed is illustrating the possibilities of the new package very neatly.\nI do have few questions/suggestions:\nWhen computing a pairwise bottleneck distance matrix between individual files, what kind of statistical analysis can be used to decide whether a distance between two genotypes/groups is statistically significant? The authors could at least put a suggestion of the follow-up analysis. That would be extremely useful for the part of a scientific community that works with mutants/introgression lines and examines root architecture to answer hypothesis driven questions.\n\nThe authors compare the classical root traits with persistent homology using PCA and non-metric multidimensional scaling and showing that the later is resulting in clear differences between fibrous and tap-root root types. I think that this difference could result in using PCA vs NMDS rather than the input. For the fair comparison, the value of two different inputs in distinguishing between two different root types, should be compared using the same method for reduction in dimensionality.\n\nAlthough the persistent homology is an alternative way of differentiating between the root types, the method is still having limited capability in distinguishing between \"mock\", \"shallow\" and \"steep\" root types (as shown in the archiDart shiny app). The same types are also not being differentiated using PCA for the \"classical\" root phenotypes. It would be very helpful if the authors state the limitations of using the homology (like - problems with implementing the root angle) and how exactly is the persistent homology complementary to the more classical analysis of root system architecture.\n\nThere is a glitch in the app when user removes the genotypes to be plotted in the tabs \"archiDRAW\" and archiHOMOLOGY\".\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "29567", "date": "12 Mar 2018", "name": "Alexander Bucksch", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nDelory et al. present an extension package for ArchiDart that allows the user to compute topological descriptors of branching plant shapes. The contribution is valuable for the community of researchers studying plant morphology. I do recognize that this was a lot of work to implement.\n\nI have several suggestions to improve the paper for the readers and the user community.\nThe paper gives overall a good intuition of how topological indices of branching structures are computed. Yet, the detail for further exploration is missing. Looking at the code and the paper, it was unclear to me how a user can change the function used to expand over the surface of the branching structure. Currently only the geodesic distance is used to extract a homology of a given branching structure. Does the code provide an interface to choose the function? In my opinion, It would be good to have a pseudo code to the paper that allows to see into the many functions used in the R implementation.\n\nThe capabilities of the software were demonstrated nicely on simulated branching structures. While the results are convincing for “perfect data” I missed a validation on noisy and incomplete data. In other words, I couldn’t find evidence on the robustness of the method and if it would translate to branching structures extracted from imaging data. It would be good to know for the reader which quality criteria apply to the input data and what are the limitations if data is partial. That way the code becomes useful for many more applications.\n\nI do appreciate the contribution a lot and know the previous papers referenced in the manuscript. However, for a new reader it is very difficult to follow all the cross-references in the paper. I would like to see a one or two sentence summary of concepts for each referenced paper that contributes to the content of the presented software paper. For example: “A nice explanation of how persistent homology can be applied to capture plant topologies is provided in 12” -> Until here it is not clear what persistent homology is, nor the concept of homology groups is explained sufficiently. The reader needs to read to more papers in order to understand H0 groups etc. In part this can be resolved by being clear in the preceding description of a graph and how the geodesic distance is measured on it. It is left to the reader to assume an unknown embedding of the graph in to a metric space. Currently, the graph is only introduced by having nodes and edges. Intuitively I would expect the distance between two nodes to be the number of edges between to nodes. Yet later on it is talked about the geodesic distance on the graph. I assume that the graph is embedded in an Euclidean space and the “straight edges” might be curves for which the curve length is taken to calculate the geodesic distance between any two nodes. Or did I got this wrong? More precision in the mathematical language would be helpful. Similar issues are present elsewhere in the manuscript whenever the explanation is referenced to another paper, e.g. to Archidart 1.0.\n\nA confusing aspect of the paper is that Archidart 1.0 has Archidart 3.0 as a follow up. Did I miss something? You state that Archidart 3.0 implements 5 new functions compared to Archidart 1.0. None of the functions presented in table 1 refer to Archidart 2.0. Should the presented version be Archidart 2.0?\n\nA very general comment: It is nothing new that geometry and topology are needed to quantify shapes. Work of Biassotti, Spagnuolo, Veltkamp etc. show that since many years. May be most closely related to the paper here is the paper Biasotti, Silvia et al. (2008)1 that discusses the interplay of geometry and topology for skeletal shape analysis. Many other papers show the same.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "30873", "date": "26 Mar 2018", "name": "Nathan D. Miller", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"archiDART v3.0: A new data analysis pipeline allowing the topological analysis of plant root systems”, presents new analysis methods available in with the archiDART platform.\nConstructive Criticisms: As is often the case with multivariate analysis, we reach for our favorite or most comfortable analysis from the myriad of linear alphabet soup. In this case, the authors choose principal component analysis (PCA) to “… determine the key traits differentiating the root systems being compared“. When using linear algebra tools for finding separation of groups based on multiple traits, the authors should consider using a discriminant style analysis such as Linear Discriminant Analysis (LDA), Multi-class LDA (mLDA), or Partial Least Squares Discriminant Analysis (PLS-DA). Unlike PCA, these methods include group structure in the objective function resulting in an answer which is closer to the authors goal.\n\nThe authors need to take care of the scale or units of the traits when using PCA. For example, if some traits yield numbers on the order of 10^3 and others yield numbers on the order of 10^-3, how would the authors expect this to affect the presented analysis? Similar care needs to be taken when using LDA/mLDA, perhaps a normalization such as z-score or min-max would overcome the problem. As the objective function for PLS-DA has z-score “built” in, it might most easily yield reasonable results.\n\nThe authors chose to use non-metric multidimensional scaling (NMDS), on the pairwise distance matrix for the persistent homology analysis and PCA for the trait-style analysis. If there a way to present a common approach between these two methodologies?\n\nComplements: Simply and most importantly, the authors meet their goal. They present a free, open source, and usable R package for persistent homology analysis and useful accompanying graphical abilities.\n\nConclusions: While a more thorough compare/contrast workup could be presented between the multivariate and persistent homology approaches, this paper does not claim to handle the task. The authors should consider softening the expectations/phrasing of PCA as a discrimination method or use a different approach.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/7-22
https://f1000research.com/articles/7-9/v1	04 Jan 18	{ "type": "Research Article", "title": "Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts", "authors": [ "Anuj Sharma", "Manish Bhomia", "Tze-Jou Yeh", "Jay Singh", "Radha K. Maheshwari", "Manish Bhomia", "Tze-Jou Yeh", "Jay Singh", "Radha K. Maheshwari" ], "abstract": "Background: Chikungunya virus (CHIKV) is a re-emerging pathogen that has caused widespread outbreaks affecting millions of people around the globe. Currently, there is no specific therapeutic drug against CHIKV, with symptomatic treatment only to manage the disease. Pi3-akt signaling has been implicated in infection of several viruses including that of CHIKV. Effect of Pi3-akt signaling inhibitors on CHIKV replication was evaluated in this study. Methods: Human primary dermal fibroblast cells were treated with inhibitors of the Pi3-akt signaling pathway. Suppression of CHIKV replication was evaluated as reduction in virus titer in cell supernatants. Effect of miltefosine (MF) on CHIKV replication was evaluated in pre and post treatment regimen. Inhibition of virus replication was determined by cell growth, virus titer and western blot. Results: Inhibition of Akt-phosphorylation significantly inhibited CHIKV replication. No effect on CHIKV replication was observed after treatment with Pi3-kinase and mTOR activation inhibitors. Further, MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens. Conclusion: Data suggests that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection. This is the first study to show inhibition of CHIKV replication by MF, and presents a case for further development of MF as an anti-CHIKV drug.", "keywords": [ "Chikungunya virus", "Akt-activation", "Pi3-akt signaling inhibitors", "FDA approved drug", "miltefosine" ], "content": "Introduction\n\nChikungunya virus (CHIKV) is an Old World alphavirus which has caused widespread outbreaks in tropical countries around the globe1–4. Lack of herd immunity combined with increased travel across the globe, and adaptation to Aedes albopictus have been suggested to contribute to the world wide spread of CHIKV5,6. The population in continental United States lack immunity to CHIKV and is at high risk of CHIKV outbreak7. CHIKV causes self-resolving febrile illness accompanied by arthralgia. Some studies have suggested that persistence of CHIKV in the joint tissue may be responsible for the long lasting arthralgia observed in the patients recovering from primary CHIKV infection8–10. There is no approved vaccine or specific antiviral drug to treat CHIKV infection. In our earlier study, Pi3-akt signaling was identified as one of the main pathways modulated by CHIKV infection11. Others have also shown modulation of Pi3-Akt signaling by CHIKV infection12–15. Therefore, in this study, the effect of inhibition of Pi3-Akt signaling on CHIKV replication was evaluated in human primary dermal fibroblast (hPDF) cells. HPDF cells were used in this study as dermal fibroblast are one of the primary targets of CHIKV infection16. Specific inhibitors of Akt-phosphorylation inhibited CHIKV replication in cell culture, suggesting Akt-phosphorylation to be important for CHIKV replication. Data mining for an FDA approved Akt-phosphorylation inhibitor, identified miltefosine (MF), which is used for treating visceral leishmaniasis17. A significant inhibition of CHIKV replication was observed in the hPDF cells treated with MF before and after the infection. This inhibition of the CHIKV replication was associated with the inhibition of Akt-phosphorylation. This is the first study to report anti-CHIKV activity of MF.\n\n\nMaterial and methods\n\nVirus: CHIKV181/25 strain of CHIKV was used in the present study and has been described earlier11. Virus was a kind gift from Dr. Michael D Parker, USAMRIID, Fredrick, MD. TC83 strain of Venezuelan equine encephalitis virus (VEEV) was used. Viruses were grown in Vero cells and sucrose gradient purified before use.\n\nInhibitors: LY294002 (Catalog # 440202), Akt inhibitor IV (Akt-IV; Catalog # 124011), Akt inhibitor VIII (Akt-VIII; Catalog # 124018), Rapamycin (Catalog # 553210), H89 (Catalog # 371963), and Miltefosine (Catalog # 475841) were purchased from EMD-Millipore (Billerica, Massachusetts 01821) and solutions were made in DMSO (catalog # KP31817) as per manufacturer’s recommendation.\n\nAntibodies: Following antibodies were purchased from Cell Signaling Technology, Inc., Danvers, MA: Akt pan (cat# 4691), and p-Akt (cat # 4060). Following secondary antibodies were used: Goat anti-mouse IgG HRP conjugated (Bio-rad, cat# 170-6517), and goat anti-rabbit IgG HRP conjugated (Bio-rad, cat# 170-6515). Anti-CHIKV monoclonal antibody (CHK-48) was a kind gift from Dr. Michael S. Diamond, Washington University School of Medicine. St. Louis, MO and was acquired under a material transfer agreement.\n\nCells: Human primary dermal fibroblast (hPDF) cells (Catalog # C-013-5C), growth media and supplements were purchased from Life Technologies (Grand Island, NY 14072). Cells were grown as per manufacture’s recommendation in culture media 106 (Catalog# M-106-500) supplemented with Low Serum Growth Supplement kit (Catalog# S-003-K).\n\nGrowth curve of CHIKV181/25 in hPDF cells: HPDF cells were plated to 80% confluence in 12 well plate. After overnight incubation, cells were infected with CHIKV181/25 at a multiplicity of infection (MOI) of 1. Cells were incubated with virus suspension for 1hr at 37°C/5% CO2. Unabsorbed virus was removed by two washes of cells with fresh media. Finally, 2 ml of fresh media was added to each well and cells were incubated at 37°C/5% CO2. Cell supernatants were collected at 6h, 12h, 24 h, 48 h and 72 h post infection from separate wells and virus titers were determined as 50% tissue culture infectivity dose (TCID50/ml).\n\nTreatment of cells with inhibitors and infection: HPDF cells were grown to 75–80% confluence and treated with various inhibitors for 6 hours. Cells were then infected with CHIKV181/25 at an MOI=1 or 0.1 and incubated at 37°C/5%CO2 for 1 hr. Unabsorbed virus was washed by rinsing cells once with fresh culture media. Cells were then incubated in fresh media containing the respective dose of inhibitors.\n\nToxicity of MF in hPDF cells: MF is a mitotic inhibitor and therefore, effect of MF on hPDF cell proliferation was tested. HPDF cells were treated with increasing doses of MF and cell growth was assesses using MTT assay. As MF was dissolved in DMSO, the concentration used for DMSO only samples was the same as the concentration of DMSO in 40 µM MF.\n\nMF pre-treatment study: Cells were treated with respective doses of MF for 4–6 h and then infected with CHIKV181/25 at an MOI=1 or 0.1. Unabsorbed virus was removed after 1hr and cells were rinsed once with fresh media. Cells were then incubated in fresh media containing respective doses of MF for 24 h post infection. DMSO and saline treated cells were used as controls. Experiment was performed in 12-well plates and drug treatment was done in triplicate wells. For protein assays, experiments were performed in 6-well plates and drug treatment was performed in triplicates. All experiments were repeated for reproducibility. A similar experimental set up was used to evaluate effect of pre-treatment of hPDF cells with MF on TC-83 replication.\n\nMF post-treatment study: Cells were first infected with CHIKV 181/25 at an MOI=1 or 0.1. Unabsorbed virus was removed and cells were incubated in fresh media without MF, except in the group where MF treatment was done at the time of infection (ATI). Cell supernatants were replaced with fresh media containing MF at pre-determined time points of ATI, 90 min, 6 h, 12 h, or 24 h post infection. Virus titers were determined in cell supernatants 24 h post-infection. Experiments were performed in 12-well plates and drug treatment was done in triplicate wells. For TC-83 study, MF treatment at only one time point of 90 min post infection was evaluated. Experiment was repeated for reproducibility.\n\nVirus titer evaluation: After 24 h post infection, cell supernatants were collected and virus titers were determine as TCID50/ml using Reed and Muench method as described before18,19. Briefly, 3000 cells were plated in each well of 96-well plate and incubated overnight at 37°C/5%CO2. Samples were serially diluted from 10-1 to 10-10 in fresh cell culture medium. 100 µl of diluted virus was added in each well, such that 8 replicates for each serial dilution were made. Plates were incubated for 3 days and cytopathic effect in cells was evaluated under a microscope. Each well was scored + or –, respectively, for the presence or absence of CPE. Cumulative percent mortality for each dilution was calculated and TCID50/ml was calculated using the following formula: 10 x [10(X) x 10(dilution at which percent mortality >50)]. Where X = [(percent mortality>50 – 50)/(percent mortality>50 – percent mortality <50)].\n\nProtein analysis: Cells were lysed with RIPA buffer (Cat# 786-489, G Biosciences, St. Louis, MO) containing phosphatase and protease inhibitors (Cat# 04693159001 and 04906845001 respectively, Roche). Samples from triplicate well for each group were pooled, and protein was estimated by Pierce BCA kits (cat# 23227, Life technologies, Grand Island, NY) as per manufacture’s protocol. Equal amount of protein was loaded on the gel (Precise 4–20% Tris-Glycine Gel, Cat# 25249, Life Technologies, Grand Island, NY). After electrophoresis, proteins were transferred on to the nitrocellulose membrane (Cat# RPN78D, GE Healthcare Life Sciences, Pittsburgh, PA). Blocking was performed using 5% non-fat dried milk in 1XTBST, and membrane was incubated with respective primary antibody at 4°C overnight over a continuous shaker. Depending on the species of the primary antibody, bands were probed either by HRP-conjugated goat anti-mouse or HRP-conjugated goat anti-rabbit antibody.\n\nAssessment of cell growth in CHIKV-infected and MF treated cells: In both the pre- and post-treated study with MF, hPDF cells in 12-well plates were left with drug and virus containing supernatant for 72 h post infection. Cell supernatants were then removed and cells were washed twice with fresh culture media. Cells were then incubated at 37°C/5% CO2 for 4 days in fresh culture media. Cells were then fixed with 0.1% crystal violet in 10% neutral buffered formalin for 90 min. Plates were washed gently under a continuous flowing tap water for 3 min. Cells were then observed under a microscope for growth. Experiments were repeated for reproducibility.\n\nStatistical Analysis: Data analysis and statistical significance was determined using GraphPad Prism 7.01 software. Significance between the drug treated and control groups was determined by Turkey’s multiple comparison test with alpha set at 0.05. A P value of ≤ 0.05 was considered significant.\n\n\nResults\n\nHPDF cells support CHIKV replication: Dermal fibroblast are the target of CHIKV infection16, therefore, replication kinetics of CHIKV181/25 was determined in hPDF. CHIKV181/25 was readily detected in the cell supernatants of hPDF cells, and reached peak titer at 24 h post infection (Figure 1). Since 24 h post infection showed peak CHIKV181/25 titers in cell supernatants, this time point was chosen to test the effect of inhibitors on virus replication.\n\nHPDF cells were infected with CHIKV 181/25 and virus titers were measured in the cell supernatants at 24 h post infection. Values presented as ± SEM. The dashed line indicates the limit of detection of assay.\n\nAkt-activation inhibitors inhibited CHIKV replication: Effect of inhibition of Pi3-kinase and mTOR activation on CHIKV replication was tested by pre-treating hPDF cells with LY294002 and rapamycin, respectively. Both LY294002 and rapamycin did not affect CHIKV replication (Supplementary figure 1A and B). Effects of inhibition of PKA and Akt-activation on CHIKV replication was tested by pre-treating hPDF cells with H89 and Akt-VIII, respectively. Both H89 and AKT-VIII significantly inhibited the CHIKV replication in hPDF cells (Figure 2). Another Akt-activation inhibitor AKT-IV, which functions separately from AKT-VIII, also inhibited CHIKV replication (Supplementary figure 2).\n\nHPDF cells were pre-treated with inhibitors for 4–6 h and infected with CHIKV181/25. Significant reduction in virus titer was observed in the cells treated with AKT-VIII and H89.\n\nMiltefosine inhibited CHIKV replication: MF is a mitotic inhibitor, due to its Akt-phosphorylation inhibition activity, and MF treatment slightly reduced hPDF cell proliferation in comparison to saline or DMSO treated controls (Figure 3A). Pre-treatment of cells with MF significantly inhibited CHIKV replication. Treatment with 20 µM and above doses of MF resulted in significant reduction in CHIKV titer in cell supernatants, however, at MOI=1, virus titers were significantly higher than the samples infected with MOI=0.1 (Figure 3B). Therefore, extent of inhibition of CHIKV replication was dependent on the initial infectious load of the virus. To test the therapeutic potential of MF, hPDF cells were infected with CHIKV181/25 at MOI=1 and then treated with 30 or 40 µM dose of MF, either at the time of infection, 90 min, 6, or 12 h post-infection (pi). A significant inhibition of CHIKV replication was observed in samples treated with MF until 6 h pi (Figure 4). Similar experiment with an MOI=0.1 showed inhibition of CHIKV replication until 12 h pi (Supplementary figure 3).\n\n(A) hPDF cells were treated with increasing doses of MF and MTT assay was done to determine the proliferation of cells. Since, MF was dissolved in dimethyl sulfoxide (DMSO), a DMSO concentration equivalent to concentration in 40µM dose was used in the assay. The control was saline treated cells. Significant effects on cell proliferation was observed in MF treated group compared to saline treated control groups. (B) hPDF Cells were treated 4–6 h with MF and infected with CHIKV181/25 with either MOI 0.1 or 1. Virus titer in the cell supernatants was measured at 24 h post infection. Turkey’s multiple comparison test using 2 way ANOVA was performed to determine significance (P < 0.05). Values are presented as ± SD. Values are presented as ± SD. P ≤ 0.05 vs control group (A & B), and ^ P <0.05 between two MOIs (B). Data is representative of two repeat experiments.\n\nhPDF cells were infected with CHIKV181/25 (MOI = 1) and then treated with 30 or 40 µM MF. Virus titers were determined in the cell supernatants at 24 h post MF treatment. A significant reduction in virus titer was observed until 6 h pi. at the time of infection (ATI). Turkey’s multiple comparison using 2 way ANOVA test was performed to determine significance. Values are presented as ± SD. * P ≤ 0.05 as compared to respective saline control group. ^ P < 0.05 between the groups indicated on graph. Data is representative of two repeat experiments.\n\nTo check if reduction in CHIKV181/25 replication was associated with inhibition of Akt-phosphorylation, a western blot was performed on the hPDF cell-lysates collected from AKT-VIII or MF treated and CHIKV181/25-infected hPDF cells. CHIKV181/25 infection increased phosphorylated-Akt (p-Akt) levels in the cells over that of the uninfected controls (Figure 5A: lanes 2 and 3). As expected, treatment with Akt-VIII reduced p-Akt levels in uninfected as well as CHIKV181/25-infected cells as compared to the respective controls, and this reduction was associated with reduction in CHIKV antigen levels (Figure 5A: lanes 10, 11). Treatment with MF diminished the p-Akt levels both in the uninfected, as well as CHIKV181/25-infected cells, and was associated with diminished CHIKV antigen levels below the detection limit of the assay (Figure 5A: lanes 4–7).\n\n(A) The cell lysates of hPDF cells infected with CHIKV181/25 were analyzed for native Akt, p-Akt, and CHIKV antigen levels by western blot analysis. Reduction in levels of CHIKV antigen was found to be concomitant with that of the reduction in the levels of p-Akt. M= ladder, C= Control uninfected cells, I = infected, and DMSO = dimethyl sulfoxide. (B) Cells were treated with corresponding doses of MF for 4–5 h and infected with CHIKV181/25 (MOI=1). After 72 h pi, virus and drug were removed and cells were washed, replenished with fresh media and incubated for 4 days. (C) Cells were infected with CHIKV181/25 (MOI=1) and treated with MF at the indicated time points post-infection. After 72 h pi, virus and drug were removed and cells were washed and replenished with fresh medium and incubated for 4 days. B and C: Cells were fixed and stained overnight with 10% neutral buffered formalin containing 0.1% crystal violet and subsequently washed gently under running tap water and observed under the microscope. Data is representative of two repeat experiments.\n\nTo determine if the inhibition of CHIKV181/25 replication by MF was complete or transient, re-activation of CHIKV181/25 replication in infected hPDF cells after the removal of MF from cell culture media was evaluated. HPDF cells were infected with CHIKV (MOI=1) and treated with MF (pre- or post-infection as described in methods section) for 72 h followed by washing of cells and replenishment with fresh growth media without MF. After 4 days a cellular monolayers were observed in CHIKV181/25-infected cells pre-treated with 30 and 40 µM of MF (Figure 5B). In CHIKV181/25 infected cells post-treated with MF, cell growth was observed in samples treated with 30 and 40 µM of MF until 6 h pi (MOI =1) (Figure 5C) and 12 h pi (MOI =0.1) (Supplementary figure 4). These results suggest that MF-induced inhibition of CHIKV181/25 was complete at ≥30µM dose of MF.\n\nTo determine if the antiviral effects of MF was specific against CHIKV, effect of MF treatment on VEEV, TC83 strain, replication was evaluated. Unlike CHIKV181/25, no inhibition of TC83 replication (MOI=0.1) was observed by pretreatment of cells with low doses of MF, and reduction in virus titer was observed only with a 40µM dose of MF. Post-infection treatment of hPDF cells with MF did not affect TC-83 replication (Supplementary figure 5), suggesting MF mediated inhibition to be more specific to CHIKV.\n\n\nConclusion\n\nCHIKV is a reemerging virus of global public health importance. Though not lethal, CHIKV causes a debilitating arthritic disease that severely affects the quality of life3,4. In the absence of specific therapeutic drugs or a vaccine, control of the CHIKV epidemic has been difficult and millions of people worldwide have been infected. Pi3-akt signaling has been shown to play an important role in viral infections. Akt-activation has been shown to be an important step during the infection of several viruses such as influenza, enterovirus and varicella zoster virus, and is suggested to help virus replication cycle by delaying or inhibiting apoptosis in the cells20–23. Phosphorylation of Akt and Akt-mediated Hsp90 activation has been shown during CHIKV replication12,13,15. Pi3-akt signaling pathways was also found to be upregulated in white blood cells isolated from the CHIKV infected patients14. Pi3-akt signaling pathway was also one of the top most pathways likely to be targeted by the modulated microRNAs following CHIKV infection11. In this study, infection with CHIKV181/25 increased p-Akt levels in hPDF cells, which is in agreement with previous observation with wild type CHIKV in BHK cells15. HPDF cells were used in this study as dermal fibroblast are the target of CHIKV infection and would provide relevant data for inhibition of CHIKV replication by drugs of interest16. Specific inhibition of Akt-phosphorylation, but not of Pi3-kinase or mTOR activation, inhibited CHIKV replication. No effect on CHIKV protein expression has been shown after treatment with specific inhibitor of Pi3-kinase activation15. Akt-VIII is a pleckstrin homology (PH) domain-dependent inhibitor of Akt-activation. The PH domain of Akt translocate Akt to the membrane where it is subsequently phosphorylated. To rule out the possibility of inhibition of CHIKV replication due to interference in translocation of Akt to the membrane, effect of AKT-IV inhibitor on CHIKV181/25 replication was tested. AKT-IV, which inhibits phosphorylation of Akt by inhibiting an upstream kinase other than Pi3-kinase, also inhibited CHIKV replication. Protein kinase A (PKA) regulate Akt-activation independent of Pi3-kinase and has been shown to play important role in viral infections24–30. Treatment of hPDF cells with H89, an inhibitor of PKA, also inhibited CHIKV181/25 replication. This observation was similar to the inhibition of hepatitis C virus by H89 reported elsewhere28. Taken together, these results suggested that phosphorylation of Akt is important for CHIKV replication.\n\nMiltefosine (MF) is an Akt-phosphorylation inhibitor, which is approved by the FDA for treating Leishmania infections in humans17. It belongs to an alkylphosphocholine drug family and is an efficient inhibitor of Akt-phosphorylation31. Therefore, as expected some reduction in hPDF cell proliferation was observed after treatment with MF. MF has also been shown to inhibit herpes simplex virus by inhibiting Akt-phosphorylation32. Significant inhibition of CHIKV181/25 replication was observed in hPDF in prophylactic, as well as therapeutic treatment regimen. The antiviral activity was observed at 20–40 µM doses of MF, which correspond to 8.1–16.3 µg/ml of MF. In clinics, MF is administered orally as 50–100 mg/kg doses, which achieve plasma concentration ranging from 24–70 µg/ml with half-life of ~7 days17. It is not possible to directly correlate in-vitro dose with in vivo doses. However, based on median plasma concentration and long half-life, MF may show anti-CHIKV activity in vivo.\n\nTo our knowledge this is the first study to report anti-CHIKV activity of MF. The study is limited by the use of attenuated strain of CHIKV, instead of the circulating wild-type strain of virus, which requires biosafety level 3 (BSL-3) containment for handling. However, we present a strong case for further evaluation of MF as anti-CHIKV drug in vivo, and against wild-type CHIKV virus.\n\n\nData availability\n\nDataset 1: Raw data underlying the results presented DOI, 10.5256/f1000research.13242.d18906333\n\n\nDisclaimer\n\nThe opinions expressed herein are that of the author(s), and are not necessarily representative of those of the Uniformed Services University of the Health Sciences (USUHS), the Department of Defense (DOD), the United States Army, Navy, Air Force, and Defense Threat Reduction Agency.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was funded by a grant from Defense Threat Reduction Agency.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe dedicate this study to our beloved mentor Dr. Radha K Maheshwari who had a great passion for research and student training. CHIKV181/25 was a kind gift from Dr. Michael D Parker, USAMRIID, Fredrick, MD.\n\n\nSupplementary material\n\nSupplementary File 1: Supplementary Figure 1–Supplementary Figure 5\n\nClick here to access the data.\n\nSupplementary Figure 1: Effect of LY294002 and Rapamycin on CHIKV181/25 replication. hPDF cells were pretreated 4–6 h with the inhibitor and infected with CHIKV181/25 at an MOI = 0.1. Virus titer in cell supernatant were measured at 24 h post infection. No effect on CHIKV replication was observed in cells pre-treated with either LY294002 (pi3k inhibitor; A) or rapamycin (mTOR inhibitor; B). Data is representative of two repeat experiment. Values are presented as ± SD.\n\nSupplementary Figure 2: Akt-IV inhibited CHIKV181/25 replication. hPDF cells were pretreated 4–6 h with Akt-IV inhibitor and infected with CHIKV181/25 at an MOI = 0.1. Virus titer in cell supernatants was measured at 24 h post infection. Turkey’s multiple comparison test using 2 way ANOVA was performed to determine significance. Values are presented as ± SD. * P ≤ 0.05 as compared to control group. ^ P ≤ 0.05 as compared to control saline treated group. Data is representative of a two repeat experiment.\n\nSupplementary Figure 3: Effect of MF treatment post-infection on CHIKV replication. A time dependent inhibition of CHIKV replication was observed in hPDF cells infected with CHIKV (MOI=0.1) and then treated with 40 µM of MF. A significant inhibition was observed until 12 h post infection. Cells that were treated after 24 h post infection did not show reduction in virus replication in cell supernatants. No virus titer was detected in the cell supernatant in the group treated with MF at the time of infection (ATI). Turkey’s multiple comparison test using 2 way ANOVA was performed to determine significance. Values are presented as ± SD. * P ≤ 0.05 as compared to control group. ^ P < 0.05 between the groups represented on the graph.\n\nSupplementary Figure 4: Effect of MF treatment on cell growth after infection with CHIKV181/25 with MOI 0.1: (A) Cells were treated with MF for 4–5 h and infected with CHIKV 181/25. After 72 h post infection, virus and drug were removed and cells were washed and replenished with fresh media and incubated for 4 days. (a) Uninfected control; (b) infected control; (c) infected + 10 µM MF; (d) infected + 20 µM MF; (e) infected + 30 µM MF; and (f) infected + 40 µM MF. (B) Cells were infected with CHIKV181/25 (MOI=0.1) and treated with MF at the time post infection as indicated (ATI= at the time of infection). After 72 h post infection, virus and drug were removed and cells were washed and replenished with fresh medium.\n\nSupplementary Figure 5: TC83 replication in hPDF cells treated with Akt-activation inhibitor or MF: (A) HPDF cells were pretreated with MF for 4–6 h and infected with TC-83 at an MOI=0.1. (B) HPDF cells were infected with TC-83 (MOI=0.1) and treated with MF 90 min after the infection. Values are presented as ± SEM. * P ≤ 0.05 as compared to DMSO treated group.\n\nSupplementary File 2: Raw data underlying supplementary figures\n\nClick here to access the data.\n\n\nReferences\n\nRezza G: Dengue and chikungunya: long-distance spread and outbreaks in naïve areas. Pathog Glob Health. 2014; 108(8): 349–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeaver SC, Forrester NL: Chikungunya: Evolutionary history and recent epidemic spread. Antiviral Res. 2015; 120: 32–39. 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Prog Mol Biol Transl Sci. 2012; 106: 223–50. PubMed Abstract \| Publisher Full Text\n\nFilippa N, Sable CL, Filloux C, et al.: Mechanism of protein kinase B activation by cyclic AMP-dependent protein kinase. Mol Cell Biol. 1999; 19(7): 4989–5000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSable CL, Filippa N, Hemmings B, et al.: cAMP stimulates protein kinase B in a Wortmannin-insensitive manner. FEBS Lett. 1997; 409(2): 253–7. PubMed Abstract \| Publisher Full Text\n\nDi Pasquale G, Chiorini JA: PKA/PrKX activity is a modulator of AAV/adenovirus interaction. EMBO J. 2003; 22(7): 1716–24. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nScherer J, Yi J, Vallee RB: PKA-dependent dynein switching from lysosomes to adenovirus: a novel form of host-virus competition. J Cell Biol. 2014; 205(2): 163–77. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarquhar MJ, Harris HJ, Diskar M, et al.: Protein kinase A-dependent step(s) in hepatitis C virus entry and infectivity. J Virol. 2008; 82(17): 8797–811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKagnoff MF, Roebuck KA: Human immunodeficiency virus type 1 (HIV-1) infection and expression in intestinal epithelial cells: role of protein kinase A and C pathways in HIV-1 transcription. J Infect Dis. 1999; 179(Suppl 3): S444–7. PubMed Abstract \| Publisher Full Text\n\nTange S, Zhou Y, Nagakui-Noguchi Y, et al.: Initiation of human astrovirus type 1 infection was blocked by inhibitors of phosphoinositide 3-kinase. Virol J. 2013; 10: 153. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRama M, Kumar NV, Balaji S: A comprehensive review of patented antileishmanial agents. Pharm Pat Anal. 2015; 4(1): 37–56. PubMed Abstract \| Publisher Full Text\n\nCheshenko N, Trepanier JB, Stefanidou M, et al.: HSV activates Akt to trigger calcium release and promote viral entry: novel candidate target for treatment and suppression. FASEB J. 2013; 27(7): 2584–99. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSharma A, Bhomia M, Yeh TJ, et al.: Dataset 1 in: Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts. F1000Research. 2017. Data Source" }	[ { "id": "29497", "date": "19 Jan 2018", "name": "Mukesh Kumar", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled \" Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts” by Sharma and colleagues evaluated the effect of AKT-activation inhibitors on Chikungunya virus replication. The manuscript is well written and easily comprehensible. The results of this work are very significant to the field and fit with the scope of this journal. The manuscript would be improved by consideration of the points below, and amendment as indicated:\n\nSimilar to MF, percent cell survival after treatment with AKT-VIII and H89 should be included.\n\nY-axis missing in Supp. Fig. 5A\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "29502", "date": "01 Feb 2018", "name": "Soma Chattopadhyay", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript by Sharma et al., entitled “Miltefosine inhibits Chikungunya virus replication in human primary dermal fibroblasts”, the authors have shown that MF, an FDA-approved Akt-inhibitor, inhibited CHIKV replication in pre- and post-infection treatment regimens And proposed that Akt-phosphorylation can be an amenable target of therapy against CHIKV infection.\nThe work is interesting and written properly. However, few things have to be corrected before consideration.\nMajor comments:\nWhat is the % of inhibition / reduction, that has been never been mentioned in case of all the inhibitors.\n\nHow all the statistical values are with one *? Fig 3 A- How 10 μM is statistically significant? Fig 3 B- How all the bar diagrams are with same p-value, actual p value should be provided here.\n\nHow the reduction of viral titer is shown at 24 hpi in Fig 3, where as in Fig 4 the significant reduction is shown only at 6 hpi and then the infection rate has been decreased.\n\nHow the current manuscript has been used as reference in this manuscript. The data is the part of this manuscript only. This cannot be referred.\n\nWhat is the need for showing saline treated cells as control has not been explained in the text.\n\nWhat are the specifications of all the inhibitors has not been explained. Why LY294002 and Rapamycin did not work, can the author give some explanation?\n\nLittle more introductions on different drugs inhibiting CHIKV replication may be mentioned in one para in the introduction.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNo\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "29844", "date": "01 Feb 2018", "name": "Rana Abdelnabi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript is well written and the methodology is clear. However I have only minor comments:\nPi3-akt signaling should be briefly introduced in the Introduction section.\n\nThe toxicity data of the used inhibitors should be included in the supplementary material.\n\nIs there any explanation why the virus controls in supplementary Figure 1B and supplementary Figure 2 are almost 1 log less than the virus controls in the rest of figures although MOI=0.1 was used in all these experiments?.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-9
https://f1000research.com/articles/6-1416/v1	10 Aug 17	{ "type": "Research Note", "title": "People who report anomalous information reception have higher dissociation symptom scores", "authors": [ "Helané Wahbeh", "Dean Radin", "Dean Radin" ], "abstract": "Background: Dissociative states exist on a continuum from nonpathological expressions, such as highway hypnosis and day-dreaming, to pathological states of derealization and depersonalization. Claims of anomalous information reception (AIR) are often dismissed as symptoms of dissociation disorder, despite other studies that show otherwise. This study examined the relationship of dissociation symptoms and AIR in a large convenience sample (n= 3,984). Methods: These secondary analyses of cross-sectional survey data were examined. The survey included demographics, the Dissociation Experience Scale Taxon, and AIR data. Summary statistics and linear and logistic regressions evaluated dissociation variables and AIR endorsement relationships with and without covariates. Results: 3023 respondents with complete data were included. Participants were mostly middle aged (51 years ± 16; range 17-96), female (70%), Caucasian (85%), college educated (88%), had an annual income over $50,000 (55%), were raised Christian (71%), and now affiliated as Spiritual but not Religious (60%). AIR ability was endorsed by 42% of participants, with their first experience starting in childhood (81%), and 53% having family members with similar experiences. The mean dissociation score was 14.4 ± 17.3 (range 0-100) for all participants and was significantly higher for AIR claimants (18.2 ± 19.3), as compared to non-claimants (11.8 ± 15.2; t = -10.3, p<0.000). In total, 11% of AIR non-endorsers and 22% of AIR endorsers had a cut-off score greater than 30 (X2 = 63.0, p=<0.000). Conclusions: Both AIR claimants and non-claimants scored lower than the clinical cutoff for dissociation, with the claimants having significantly higher scores. Future studies comparing AIR claimants versus non-claimants may benefit by incorporating comprehensive dissociation symptom measurement, as well as their effects on the person’s functionality, to discern the pathological versus non-pathological nature of purported AIR experiences.", "keywords": [ "Dissociation", "Anomalous information reception" ], "content": "Introduction\n\nDissociation is conceptualized as the disruption to usually integrated functions of consciousness, memory, identity or perception of the environment1. Dissociative Identity Disorder is defined as a personality disorder, when two or more distinct identities or personalities are present, each with its own pattern of perceiving, relating to and thinking about the environment and self. The core clinical symptoms of dissociative disorders include amnesia, depersonalization, derealization, identity confusion and identity alteration. Dissociative states are prevalent in other psychiatric disorders, such as PTSD2,3, and are more prevalent in younger nonclinical populations3. Dissociative states exist on a continuum4–6, from nonpathological expressions, such as highway hypnosis and day-dreaming, to pathological states of derealization (surrealness), and depersonalization (absence of identity)7. Almost half of United States adults have experienced a dissociative episode in their lives3.\n\nA widespread belief possibly related to dissociation is the idea that it is possible to communicate with deceased individuals; people who report such experiences are called “mediums”8. A survey of 18,607 people in thirteen European countries found that 25% reported contact with the dead9. Some empirical literature suggests that in some cases the information obtained is accurate even under double-blind conditions10–13. Claims of such abilities are often considered to be symptoms of dissociation disorders5,14,15, despite the fact that pathological dissociation studies have not systematically indicated increased prevalence in people who maintain these claims compared to control groups or the general population16,17.\n\nThis study’s aim was to examine the relationship between self-report dissociation symptoms and anomalous information reception (AIR) about deceased humans in a large convenience sample of surveyed adults. We hypothesized that the prevalence of pathological dissociative symptoms in people who claim these purported abilities would be the same as in those who do not maintain such claims.\n\n\nMethods\n\nThese analyses were performed on data collected during a larger research study approved by the Institute of Noetic Sciences (IONS) Institutional Review Board (approval number, wahh_2016_01). A survey was administered through SurveyMonkey.com with HIPAA compliant methods. Participants were recruited through the IONS Facebook page, IONS mailing lists, including the IONS membership list, and the IONS community networks.\n\nThe survey (Supplementary File 1) began with the study’s purpose and informed consent details. Date and country of birth, race, education, and childhood and current spiritual/religious affiliation and education were collected. Gender was collected on a subsample of participants. Participants indicated if they had experienced AIR or “mediumship,” defined as the “ability to mediate communication between spirits of the dead and the living or the empathic ability to feel the presence and energies of spirits,” age of onset (if applicable), and family history of AIR.\n\nThe Dissociation Experiences Scale Taxon (DES-T)18 distinguishes pathological dissociation with a cutoff score of 30, which captures 87% positive predictive value (Cronbach Alpha of 0.75)19,20. Respondents select a percent frequency for eight dissociative symptoms. The DES-T results in two variables: a continuous variable calculated from the mean of the eight items; and a binary variable based on the >30 cutoff score18.\n\nCategorical variable percentages were calculated and presented qualitatively. Means, standard deviations and ranges of continuous variables were calculated. Covariates included gender, age, race, education, income, childhood spirituality and current spirituality, family history, and age of the claimed ability onset. Missing values were randomly distributed except for gender. T-test and chi-square tests evaluated relationships among the demographic variables. Linear and logistic regressions evaluated dissociation variables and AIR endorsement relationships. Statistics were performed with Stata 12.0.\n\n\nResults\n\nIn total, 3984 participants took the survey from May 4, 2016 to June 7, 2017. Participants were not required to complete all fields and thus only data from 3023 participants who answered the “AIR” question (question 49 of the survey) and completed the DES-T (question 75) were included. Most participants were from the United States (62.6%) followed by the United Kingdom (7.7%) and then Canada (6.3%), and the remaining participants represented thirteen other countries. Participants were mostly middle aged (51 years ± 16; range 17-96), female (70%), Caucasian (85%), college educated (88%), had an annual income over $50,000 (55%), were raised Christian (71%), and now affiliated as Spiritual but not Religious (60%; Table 1). Gender, race and current spiritual/religious affiliation were different between people who did and did not endorse AIR.\n\nMean ± standard deviation; t, Student’s two-sample t-test statistic; X2, chi-square statistic; p, probability.\n\nAIR ability was endorsed by 42% of participants, with their first experience starting in childhood (81%), and 53% having family members with similar experiences. The mean DES-T score was 14.4 ± 17.3 (range 0-100) for all participants and was significantly higher for AIR claimants (18.2 ± 19.3) as compared to non-claimants (11.8 ± 15.2; t = -10.3, p<0.000; Table 2). A DES-T continuous variable linear regression model including all covariates found only race and education to be significant. Repeating the model with these covariates resulted in a highly significant DES-T difference between groups (F (3, 2947) = 73.2, p<0.0000). For the DES-T binary cutoff score, 11% of AIR non-endorsers and 22% of AIR endorsers had a cut-off score greater than 30 (X2 = 63.0, p<0.000). These values are significantly different with education (> college) and income (>$50,000) as covariates in a logistic regression (LR X2 = 99.12, p< 0.0000).\n\nData are presented as the mean ± standard deviation. DES-T, Dissociation Experiences Scale Taxon; t - Student’s two-sample t-test statistic; p, probability.\n\n\nDiscussion\n\nIn total, 42% of participants endorsed AIR experiences in this convenience sample, similar to other prevalence belief studies9,21,22. The overall dissociation mean score for AIR respondents fell below the clinical cutoff for pathological dissociation despite being higher than and different to non-endorser scores. Much debate exists for the use of cutoff scores18,23. Notably, the top five endorsed DES-T items were consistent with an AIR experience. Also, our total samples grand mean DES-T score was higher than observed in random general population samples19. This likely reflects the convenience sampling method for this survey, which reduces the generality of these findings. This outcome also does not clarify if AIR endorsers with high DES-T scores have the five core clinical symptoms of dissociation. Future studies comparing AIR claimants versus non-claimants may benefit by incorporating comprehensive dissociation symptom measurement, as well as their effects on the person’s functionality.\n\n\nData availability\n\nDataset 1: Dissociation symptoms for those with and without self-report anomalous information reception. DT# are the Dissociation Experience Scale Taxon items. doi, 10.5256/f1000research.12019.d17135224", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by FUNDAÇÃO BIAL (grant number No. 257/14).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank Amira Sagher, Leena Michel and the Institute of Noetic Sciences.\n\n\nSupplementary material\n\nSupplementary File 1: Survey on genetics of psychic ability.\n\nClick here to access the data.\n\n\nReferences\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, (5th ed.). Washington DC: American Psychiatric Publishing Inc, 2013. Publisher Full Text\n\nCoons PM: Confirmation of childhood abuse in child and adolescent cases of multiple personality disorder and dissociative disorder not otherwise specified. J Nerv Ment Dis. 1994; 182(8): 461–464. PubMed Abstract \| Publisher Full Text\n\nRoss CA, Joshi S, Currie R: Dissociative experiences in the general population. Am J Psychiatry. 1990; 147(11): 1547–1552. PubMed Abstract \| Publisher Full Text\n\nKihlstrom JF: Dissociative disorders. Annu Rev Clin Psychol. 2005; 1: 227–253. PubMed Abstract \| Publisher Full Text\n\nSeligman R, Kirmayer LJ: Dissociative experience and cultural neuroscience: narrative, metaphor and mechanism. Cult Med Psychiatry. 2008; 32(1): 31–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRuchsow M, Herrnberger B, Beschoner P, et al.: Error processing in major depressive disorder: evidence from event-related potentials. J Psychiatr Res. 2006; 40(1): 37–46. PubMed Abstract \| Publisher Full Text\n\nStolovy T, Lev-Wiesel R, Witztum E: Dissociation: adjustment or distress? Dissociative phenomena, absorption and quality of life among Israeli women who practice channeling compared to women with similar traumatic history. J Relig Health. 2015; 54(3): 1040–1051. PubMed Abstract \| Publisher Full Text\n\nHaraldsson E: Popular psychology, belief in life after death and reincarnation in the Nordic countries, Western and Eastern Europe. Nordic Psychology. 2006; 58(2): 171–180. Publisher Full Text\n\nHaraldsson E, Houtkooper JM: Psychic experiences in the multinational human values study: Who reports them. Journal of the American Society for Psychical Research. 1991; 85(2): 145–165. Reference Source\n\nDelorme A, Beischel J, Michel L, et al.: Electrocortical activity associated with subjective communication with the deceased. Front Psychol. 2013; 4: 834. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDelorme A, Pierce A, Michel L, et al.: Prediction of Mortality Based on Facial Characteristics. Front Hum Neurosci. 2016; 10: 173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeischel J, Boccuzzi M, Biuso M, et al.: Anomalous information reception by research mediums under blinded conditions II: replication and extension. Explore (NY). 2015; 11(2): 136–142. PubMed Abstract \| Publisher Full Text\n\nBeischel J, Schwartz GE: Anomalous information reception by research mediums demonstrated using a novel triple-blind protocol. Explore (NY). 2007; 3(1): 23–27. PubMed Abstract \| Publisher Full Text\n\nCastillo RJ: Trance, functional psychosis, and culture. Psychiatry. 2003; 66(1): 9–21. PubMed Abstract \| Publisher Full Text\n\nSeligman R: Distress, dissociation, and embodied experience: Reconsidering the pathways to mediumship and mental health. Ethos. 2005; 33(1): 71–99. Publisher Full Text\n\nRoxburgh EC, Roe CA: A survey of dissociation, boundary-thinness, and psychological wellbeing in spiritualist mental mediumship. The Journal of Parapsychology. 2011; 75(2): 279–299. Reference Source\n\nNegro PJ Jr, Palladino-Negro P, Louzã MR: Do religious mediumship dissociative experiences conform to the sociocognitive theory of dissociation? J Trauma Dissociation. 2002; 3(1): 51–73. Publisher Full Text\n\nWaller NG, Ross CA: The prevalence and biometric structure of pathological dissociation in the general population: taxometric and behavior genetic findings. J Abnorm Psychol. 1997; 106(4): 499–510. PubMed Abstract \| Publisher Full Text\n\nWaller N, Putnam FW, Carlson EB: Types of dissociation and dissociative types: A taxometric analysis of dissociative experiences. Psychol Methods. 1996; 1(3): 300–321. Publisher Full Text\n\nZingrone NL, Alvarado CS: The Dissociative Experiences Scale-II: Descriptive Statistics, Factor Analysis, and Frequency of Experiences. Imagin Cogn Pers. 2001; 21(2): 145–157. Publisher Full Text\n\nGreeley A: Mysticism goes mainstream. American Health. 1987; 7: 47–49. Reference Source\n\nCastro M, Burrows R, Wooffitt R: The paranormal is (still) normal: The sociological implications of a survey of paranormal experiences in Great Britain. Sociol Res Online. 2014; 19(3): 16. Publisher Full Text\n\nLeavitt F: Dissociative Experiences Scale Taxon and measurement of dissociative pathology: Does the taxon add to an understanding of dissociation and its associated pathologies? J Clin Psychol Med Settings. 1999; 6(4): 427–440. Publisher Full Text\n\nWahbeh H, Radin D: Dataset 1 in: People who report anomalous information reception have higher dissociation symptom scores. F1000Research. 2017. Data Source" }	[ { "id": "24906", "date": "21 Aug 2017", "name": "Etzel Cardeña", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is a good example of why the F1000Research model is so bad... The manuscript is poorly written, does not show a good grasp of the relevant literature or that a good literature search was conducted, misrepresents some of its references, and very probably has an important mistake in a Table or its analyses. All of these issues could have been solved during the regular peer review process so that at the end the only publicly available version would have been an adequate one.\nAlthough it is a short paper, it would take me too long to list all of the problems in it, so I will just mention 2-3 examples per problematic issue:\nPoor writing: From the abstract: a) \"symptoms of [a] dissociation [dissociative] disorder\". b) \"Both AIR claimants and non-claimants scored lower than the clinical cutoff\" [despite the previous sentence in the abstract mentioning that percentages of both groups had scored above the cutoff. c) \"incorporating [a] comprehensive dissociative symptom measurement, as well as their effects on the person's functionality\" [grammatical number is inconsistent, besides the fact that \"measurement[s]\" would not have an effect on functionality.\n\nInadequate coverage of the literature: a) There have been various recent studies specifically evaluating possible psychopathology in people reporting anomalous experiences (in general) and spirit possession/mediumship (in particular), yet only very few are listed in the Reference section. b) Contrary to what the authors write that \"Claims of such abilities are often considered to be symptoms of dissociation [dissociative] disorders\", yet both the anthropological literature and, more relevant in this case, the Diagnostic and Statistical Manual taxonomy, ever since its 4th edition, has specifically required that clinically significant levels of distress or dysfunction be present to consider a dissociative manifestation pathological.\n\nMisrepresentations of cited literature: a) A paper by Rebecca Seligman is used to support the above quotation that mediumship abilities are often considered to be symptoms of dissociation, yet she specifically states that \"dissociation is not a pathological experience, but rather a therapeutic mechanism\", along the lines of what others in anthopology and psychology have written. b) \"Almost half of United States adults...\", yet this study was conducted in Winnipeg, Canada.\n\nStatistical issues: a) In Table 1, a 3% difference (87 vs 84%) is reported as significant at the minus .05 level, yet an almost identical difference with about the same number of participants (87 vs 89%) is reported as non-significant. I very much doubt that both statements, particularly the first one, are accurate. b) There are multiple references to probability values = 0 or less than 0, but of course it goes against inferential statistics to state that instead of, for example, less than .001, or whatever.\n\nIs the work clearly and accurately presented and does it cite the current literature? No\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? No\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3099", "date": "23 Oct 2017", "name": "Helané Wahbeh", "role": "Author Response", "response": "Response to Reviewer #1 \"This paper is a good example.....so that at the end the only publicly available version would have been an adequate one.\" -Thank you for you taking the time to review our paper and for your thoughtful comments. Yes, we agree that through the traditional peer-review process, the final public version is superior to initial versions. We appreciate the transparent nature of F1000 review process as a way to reduce bias in publishing. \"Poor writing\" -Thank you for highlighting writing errors in our manuscripts. We have corrected the highlighted grammatical errors and unclear wording. We have also reviewed the entire paper for other writing errors. \"Inadequate coverage of the literature\" -We agree that the paper does not fully examine the literature related to this topic. The reason for this is the word count limits of a short article (1000 words). We have included additional references and attempted to succinctly include a broader understanding of the current literature. \"Misrepresentations of cited literature\" a) A paper by Rebecca Seligman - Rebecca Seligman also states “Medical approaches to the question have implicated psychological disturbance as a motivational factor for some, yet they fail to explain how and why some psychological disturbances, in some individuals, come to be expressed as spirit-possession mediumship.” She also has a very nice discussion in the section “MEDIUMSHIP AND MENTAL ILLNESS REVISITED” on mediumship being considered a pathology. Because of the word limitations, we did not highlight the specific sections of her paper that refer to these issues but allow the reader to read the paper to appreciate the full scope of the discussion of mediumship and mental illness. b) \"Almost half of United States adults...\", yet this study was conducted in Winnipeg, Canada. -This reference has been corrected. \"Statistical issues\" a) In Table 1, a 3% difference (87 vs 84%) ..... I very much doubt that both statements, -particularly the first one, are accurate.\" -These statistics have been double checked and they are accurately represented. We have also included a conservative Bonferroni multiple comparisons correction. b) There are multiple references to probability values = 0 or less than 0, but of course it goes against inferential statistics to state that instead of, for example, less than .001, or whatever. -The p statistic reporting has been corrected." } ] } ]	1	https://f1000research.com/articles/6-1416
https://f1000research.com/articles/7-6/v1	03 Jan 18	{ "type": "Case Report", "title": "Case Report: Case report on multiple extradural thoracic lesions with myelopathy as the clinical presentation in a systemic sarcoidosis- another tale of a lurking entity", "authors": [ "Sunil Munakomi" ], "abstract": "Herein, we report a rare case study of a sarcoidosis presenting with the features of compressive myelopathy. There were multiple extra-dural lesions in the thoracic region. Computerized Tomography (CT) of the chest revealed fibrotic changes with a pleural based nodular lesion in the right lung. The patient underwent laminectomy and partial excision of both the lesions. The histology revealed presence of non-caseating granulomas. The patient made a good recovery following adjuvant medical management with steroid and Methotrexate. Repeat CT scan of the chest also confirmed good resolution in the size of the pleural based nodule.", "keywords": [ "neuro-sarcoidosis", "myelopathy" ], "content": "Introduction\n\nThe involvement of the spine due to sarcoidosis is a rare occurrence1. The involvement of an extradural region, and its presentation with features of compressive myelopathy, is an even more uncommon epi-phenomenon2. Herein, we present a case study of a 50 year old male patient, who had collaborative radiological and histological evidence of an extradural spinal sarcoidosis with pulmonary involvement. The patient showed good improvement following therapy with steroids and Methotrexate. This highlights the clinical implication of keeping such entities in the differential diagnosis in patients presenting with myelopathy for better therapeutic benefits. Though a rarity, sarcoidosis still remains a lurking entity with higher tendency for multisystem involvement.\n\n\nCase study\n\nA 50-year old male farmer, presented for the first visit to our spine clinic in December 2017 with a history of slowly progressing weakness of his bilateral lower limbs over last 4 months. Due to financial restrain, he decided not seeking medical help for his symptoms. However, in recent weeks, he was not even able to stand on his own, thereby restricting him from carrying out even most basic activities of his daily life. The patient declared no history of trauma or fall injuries. There was no fever, chronic cough, significant weight loss, or swellings elsewhere in the body, or any bladder or bowel symptoms. He had no relevant history of close contact with patients having Tuberculosis, which is among the major causes of myelopathy in our context. There was no relevant past medical or surgical illnesses, or family history of any malignancies or similar ailments.\n\nThe patient was well built, conscious and well oriented to time, place and person. There was no pallor or lymphadenopathy. His vision, as well as all cranial nerves was intact. Spinal examination revealed presence of sensory loss below T7 with exaggerated knee reflexes and up-going Babinski sign suggesting myelopathy. Vibration sense was disturbed from the D4 spinous process. Systemic examination was clinically normal. Urgent Magnetic Resonance Imaging (MRI) of the spine revealed the presence of multiple enhancing extradural lesions compressing the cord in the D3-D4 and D9-D10 thoracic region (Figure 1). There were posteriorly based lesions with iso-hypointense in the T1 image, hypointense in the T2 image and homogenously enhancing following contrast administration.\n\nWe had an initial differential diagnosis of multiple metastatic lesions to the spine with features of compressive myelopathy. Ultrasound of the abdomen was normal. Computerized Tomography (CT) of the chest, however, revealed multiple fibrotic changes in the right upper lung fields with a posteriorly based pleural nodule (Figure 2). Since the patient was having compressive lesions with deteriorating neurological status, we advised the patient on the urgent need of surgical decompression and a biopsy of the lesions in planning its subsequent management.\n\nAfter receiving written consent for the surgery, the patient underwent thoracic laminectomy. There were firm and densely adherent dural based lesions. Adequate bony decompression and subtotal resection was carried out, and then sent for histopathological study. Histology was negative for any malignant cells. It revealed presence of lymphocytes with few scattered non-caseating granulomas (Figure 3). Acid Fast Staining (AFB) was negative. Serum Angiotensin Converting Enzyme (ACE) assays was marginally high. MRI screening of the brain was normal. We advised the patient of the significance of having a Flu-deoxy-glucose Positron Emission Tomography (FDG-PET) scan, allowing more accurate confirmation of the diagnosis. However, the financial aspect again proved to be a limiting issue. Following a thorough examination with the radio-pathological findings, the final diagnosis of a pulmonary sarcoidosis with extradural spinal involvement was made.\n\nThe patient was started on Oral Prednisolone (tapering dose starting from 40mg/day) and weekly Methotrexate (7.5mg) therapy. Since then, the patient has good neurological improvement, being able to walk independently with a walking stick within 10 days. The patient could walk independently in a follow up visit at 4 weeks. Repeat CT scans of his chest revealed good resolution on the pleural based lesion as well (Figure 4). We advised the patient to slowly reduce the drugs within the next 4 months. He was advised about regular follow up for periodic neurological and pulmonary examinations to determine early recurrence or progression of the disease.\n\n\nDiscussion\n\nThe spine is affected in only 1% of patients with sarcoidosis1. As such, epidural involvement presenting as a myelopathy is even a rare entity2. Cerebral spinal fluid (CSF) ACE level is not reliable enough in coming to the diagnosis in an isolated neuro-sarcoidosis without systemic involvement3.\n\nJunger et al. formatted imaging characteristics to diagnose spinal medullary sarcoidosis with findings of linear streaks, then the parenchymal enhancement followed by medullary changes with reduction of edema, and finally the cord atrophy4. However, the most common finding seen in cases with neuro-sarcoidosis is the lepto-meningeal enhancement5. FDG-PET scans may be a valuable adjunct in assessing extra-cranial lesions for biopsy in making a confirmative diagnosis6. However, availability and its high costs are the major limiting factors for its regular appliance. Histological finding of a non-caseating granulomas are conclusive7.\n\nIn order to facilitate the diagnosis, and thereby the subsequent management in cases with neuro-sarcoidosis, a diagnostic algorithm has been formulated8.\n\nManagement with use of steroids and immunosuppressant (as a steroid sparing agent), both blocking the TNF, is the current therapeutic recommendation for systemic involvement9.\n\nDue to the paucity of such cases, difficulty in assessing their clinical behavior, and high propensity for recurrence and progression, it is prudent for a prolong follow up with clinical and radiological examinations10.\n\nClinical ascertainment of such cases with neuro-sarcoidosis still remains a major challenge5.\n\nOur case was unique in the sense that myelopathy was the presenting feature in a spinal sarcoidosis, with pulmonary involvement as well. There were multiple extradural lesions in the thoracic region causing the compression. Urgent spinal decompression and histological study of such lesions followed by medical therapy with steroid and Methotrexate is advocated in such a case2. Compressive myelopathy was the presenting feature in 19% of cases in a cohort study of 54 patients with neuro-sarcoidosis5. Steroid with various immune-suppressants were used in that study, with steroid and Methotrexate utilized in 15% of the cases5.\n\n\nConclusion\n\nThis case highlights the clinical implications of keeping sarcoidosis in the differential diagnosis in patients presenting with features of myelopathy but having evidence of multisystem involvement. It is advisable for those patients to have prolonged follow ups so that an early assessment of any signs of recurrence or progression could be established.\n\n\nConsent\n\nWritten consent was obtained from the patient regarding the publication of the clinical data and the relevant radiological images.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBradley DA, Lower EE, Baughman RP: Diagnosis and management of spinal cord sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis. 2006; 23(1): 58–65. PubMed Abstract\n\nWeissman MN, Lange R, Kelley C, et al.: Intraspinal epidural sarcoidosis: case report. Neurosurgery. 1996; 39(1): 179–181. PubMed Abstract \| Publisher Full Text\n\nKhoury J, Wellik KE, Demaerschalk BM, et al.: Cerebrospinal fluid angiotensin-converting enzyme for diagnosis of central nervous system sarcoidosis. Neurologist. 2009; 15(2): 108–11. PubMed Abstract \| Publisher Full Text\n\nJunger SS, Stern BJ, Levine SR, et al.: Intramedullary spinal sarcoidosis: clinical and magnetic resonance imaging characteristics. Neurology. 1993; 43(2): 333–337. PubMed Abstract \| Publisher Full Text\n\nPawate S, Moses H, Sriram S: Presentations and outcomes of neurosarcoidosis: a study of 54 cases. QJM. 2009; 102(7): 449–460. PubMed Abstract \| Publisher Full Text\n\nBolat S, Berding G, Dengler R, et al.: Fluorodeoxyglucose positron emission tomography (FDG-PET) is useful in the diagnosis of neurosarcoidosis. J Neurol Sci. 2009; 287(1–2): 257–259. PubMed Abstract \| Publisher Full Text\n\nNowak DA, Widenka DC: Neurosarcoidosis: a review of its intracranial manifestation. J Neurol. 2001; 248(5): 363–372. PubMed Abstract \| Publisher Full Text\n\nSaleh S, Saw C, Marzouk K, et al.: Sarcoidosis of the spinal cord: literature review and report of eight cases. J Natl Med Assoc. 2006; 98(6): 965–976. PubMed Abstract \| Free Full Text\n\nBaughman RP, Lower EE, du Bois RM: Sarcoidosis. Lancet. 2003; 361(9363): 1111–8. PubMed Abstract \| Publisher Full Text\n\nLury KM, Smith JK, Matheus MG, et al.: Neurosarcoidosis--review of imaging findings. Semin Roentgenol. 2004; 39(4): 495–504. PubMed Abstract \| Publisher Full Text" }	[ { "id": "30617", "date": "22 Feb 2018", "name": "Walid Radwan", "expertise": [ "Reviewer Expertise Neurosurgery", "spine surgery", "spine deformity", "brain tumors", "neurosarcoidosis", "glioblastoma multiforme" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article is a case report of a patient presenting with myelopathy and found to have neurosarcoidosis. Relevant history and examination is provided. In addition, imaging findings are consistent and described accurately.\nHowever, with regards to recommending FDG-PET after obtaining pathological specimen it is not necessary and is not a cost-effective method in the management of this diagnosis. The diagnosis is confirmed on pathology which was obtained in this patient.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "29430", "date": "25 Jun 2018", "name": "Sachin Anil Borkar", "expertise": [ "Reviewer Expertise neurosurgery", "spine surgery", "skull base surgery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIt is a well written manuscript regarding a rare entity.\n\nIt is a well-illustrated case of a rare disease presenting in a rare fashion.\nIt should be kept in the differential diagnosis in TB endemic area presenting with myelopathy.\n\nThe manuscript can be accepted.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "34638", "date": "28 Jun 2018", "name": "Alfredo De Giorgi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article reports an uncommon case report of a patient presenting with extradural thoracic lesions related to neurosarcoidosis.\nThe case is appropriately presented.\nI recommend, if possible, to add a mini Literature review of previous cases for a better evaluation of the originality and rarity of the case reported.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/7-6
https://f1000research.com/articles/7-1/v1	02 Jan 18	{ "type": "Opinion Article", "title": "Should safety of the flu vaccine for cancer patients be reexamined?", "authors": [ "Slobodan Paessler", "Veljko Veljkovic", "Slobodan Paessler" ], "abstract": "Seasonal flu vaccine is recommended as the best protection for cancer patients against influenza infection. Recent in silico and experimental data suggest that antibodies elicited with influenza vaccine could activate bradykinin receptor B2-associated signaling pathway, which is also involved in cell proliferation and migration of tumor cells. These results point to an urgent need for the reexamination of safety of influenza vaccine(s) in cancer patients.", "keywords": [ "breast cancer", "influenza", "vaccine" ], "content": "\n\nCancer and cancer treatment can weaken the immune system making cancer patients particularly vulnerable to complications of infections, especially from acute respiratory infections such as influenza. For this reason, seasonal flu vaccine is recommended for most people with cancer and cancer survivors as the best protection against the influenza infection1.\n\nSeveral epidemiological studies have suggested that vaccination against seasonal influenza virus(es) significantly reduces the risk of major cardio-vascular events in patients with acute coronary syndrome or coronary artery disease2,3. The first clinical study on the effect of influenza vaccination after heart attack on future cardiovascular prognosis is underway. The molecular mechanism underlying protection of flu vaccine against cardiovascular diseases is unknown; however, this phenomenon is independent from vaccine efficacy against influenza A infections. Recently, it was suggested that the vaccine against influenza A viruses could elicit agonistic antibodies for bradykinin receptor B2 (BKB2R), which activates a BRB2R-associated signaling pathway that may contribute to the protection against cardiovascular diseases4. Moreover, it has been established that antibody activation of BKBR2 is possible, with all biological activities associated with it5. Recently, a monoclonal antibody with agonistic BKBR2 activity as well as anti-influenza A activity has been patented for multiple purposes6. Taken together, these data support the hypothesis of “molecular mimicry” between BKBR2 and hemagglutinin (HA) of influenza A viruses that may allow for generation of cross reactive antibodies.\n\nIn addition, it was found that levels of kinins in biological fluids of cancer patients are increased and that activation of kinin receptors expressed on cancer cells produces an increase in cell proliferation and migration of tumor cells [reviewed in Ref. 7]. Additionally, it has been demonstrated that tumor growth is increased by stimulation of kinin receptors expressed on other cells within the tumor microenvironment7, and that bradykinin and its receptors are involved in pathogenesis of numerous common cancers (gastric8, hepatocellular9, brain10, bladder11, renal12, prostate13 and breast14). These data point out that, because of possible activation of BKB2R with antibodies elicited by influenza vaccine, safety of this vaccine in cancer patients is an important issue.\n\nScreening of the clinical trials database (clinicaltrial.gov) for trials that investigated safety of the influenza vaccine in cancer patients with solid tumors (literature data connect pathogenesis of this type of tumors with BKB2R-pathway) revealed only five completed studies (Table 1). Patients were monitored in the period between 21 days and 6 months following vaccination and results were released only for one study (NCT01666782 in Table 1) for the monitoring time frame of 21 days. These short-term studies suggest that influenza vaccination is effective and safe in cancer patients in general1,15–17. However, long-term studies might be needed to test the hypothesis that if antibodies elicited by the seasonal flu vaccine may contribute to activation of BKB2R in cancer patients with potentially far-reaching consequences.\n\nIn conclusion, previously published results suggest that influenza vaccines could produce antibodies with BKB2R-agonistic activity. On the other hand, experimental and clinical data showed that activation of the bradykinin pathways plays an important role in pathogenesis of several common solid tumors. All these data suggest that until the role of the influenza vaccine in activation of BKB2R is clarified, vaccination of cancer patients against flu should be taken with some caution, and vaccines need to be monitored beyond the flu season. This especially concerns children with cancer, who represent the most vulnerable population of oncology patients. In addition, previous in silico analysis of informational properties of BKB2R and HAs from different influenza A viruses suggested that flu vaccines are not equally efficient in production of agonistic antibodies for BKB2R4. This opens the possibility for selection of antigens with low crossreactivity with BKB2R and design influenza vaccines incapable of inducing production of cross-reactive antibodies for safer use in cancer patients.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nEliakim-Raz N, Vinograd I, Zalmanovici Trestioreanu A, et al.: Influenza vaccines in immunosuppressed adults with cancer. Cohrane Database Syst Rev. 2013; (10): CD008983. PubMed Abstract \| Publisher Full Text\n\nUdell JA, Zawi R, Bhatt DL, et al.: Association between influenza vaccination and cardiovascular outcomes in high-risk patients: a meta-analysis. JAMA. 2013; 310(16): 1711–1720. PubMed Abstract \| Publisher Full Text\n\nBarnes M, Heywood AE, Mahimbo A, et al.: Acute myocardial infarction and influenza: a meta-analysis of case-control studies. Heart. 2015; 101(21): 1738–1747. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVeljkovic V, Glisic S, Veljkovic N, et al.: Influenza vaccine as prevention for cardiovascular diseases: possible molecular mechanism. Vaccine. 2014; 32(48): 6569–6575. PubMed Abstract \| Publisher Full Text\n\nWilliams MS, Charles M: A novel bradykinin-2 receptor agonist antibody that improves diabetic and cardiovascular endpoints. Circulation Res. 2012; e379–e387.\n\nWilliams MS, Charles M: Antibody-bradykinin B2 receptor (BKB2R) monoclonal antibody. US Patent Appl. 2014; 20140017242. Reference Source\n\nda Costa PL, Sirois P, Tannock IF, et al.: The role of kinin receptors in cancer and therapeutic opportunities. Cancer Lett. 2014; 345(1): 27–38. PubMed Abstract \| Publisher Full Text\n\nWang G, Sun J, Liu G, et al.: Bradykinin Promotes Cell Proliferation, Migration, Invasion, and Tumor Growth of Gastric Cancer Through ERK Signaling Pathway. J Cell Biochem. 2017; 118(12): 4444–4453. PubMed Abstract \| Publisher Full Text\n\nChen Y, Yu Y, Sun S, et al.: Bradykinin promotes migration and invasion of hepatocellular carcinoma cells through TRPM7 and MMP2. Exp Cell Res. 2016; 349(1): 68–76. PubMed Abstract \| Publisher Full Text\n\nSeifert S, Sontheimer H: Bradykinin enhances invasion of malignant glioma into the brain parenchyma by inducing cells to undergo amoeboid migration. J Physiol. 2014; 592(22): 5109–5127. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSgnaolin V, Pereira TC, Bogo MR, et al.: Functional and molecular characterization of kinin B1 and B2 receptors in human bladder cancer: implication of the PI3Kγ pathway. Invest New Drugs. 2013; 31(4): 812–822. PubMed Abstract \| Publisher Full Text\n\nKramarenko II, Morinelli TA, Bunni MA, et al.: The bradykinin B2 receptor induces multiple cellular responses leading to the proliferation of human renal carcinoma cell lines. Cancer Manag Res. 2012; 4: 195–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYu HS, Lin TH, Tang CH: Bradykinin enhances cell migration in human prostate cancer cells through B2 receptor/PKCδ/c-Src dependent signaling pathway. Prostate. 2013; 73(1): 89–100. PubMed Abstract \| Publisher Full Text\n\nGreco S, Muscella A, Elia MG, et al.: Mitogenic signalling by B2 bradykinin receptor in epithelial breast cells. J Cell Physiol. 2004; 201(1): 84–96. PubMed Abstract \| Publisher Full Text\n\nWumkes ML, van der Velden AM, Los M, et al.: Serum antibody response to influenza virus vaccination during chemotherapy treatment in adult patients with solid tumours. Vaccine. 2013; 31(52): 6177–6184. PubMed Abstract \| Publisher Full Text\n\nShehata MA, Karim NA: Influenza vaccination in cancer patients undergoing systemic therapy. Clin Med Insights Oncol. 2014; 8: 57–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVollaard A, Schreuder I, Slok-Raijmakers L, et al.: Influenza vaccination in adult patients with solid tumours treated with chemotherapy. Eur J Cancer. 2017; 76: 134–143. PubMed Abstract \| Publisher Full Text" }	[ { "id": "29869", "date": "31 Jan 2018", "name": "Emmanuele Crespan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this article, the authors suggest that flu vaccine could elicit antibodies against BKB2R. This, in turn, would lead to BKB2R activation and, therefore, activate/reinforce pro-tumoral pathways. The suggestion for the possible generation of anti-BKB2R antibodies comes from an in silico work of the authors and is supported by the observation that BKB2R monoclonal antibodies inhibit influenza virus replication in cell lines (data from a patent). These considerations are not solid enough to advise against the flu vaccination of cancer patients. Moreover, the authors should clearly state in the text that there are no experimental data yet supporting their suggestion for anti-BKB2R antibodies production upon flu vaccination.\nThe authors suggest that BKB2R antibodies would activate the receptor. However, reasoning in the same way, these antibodies should also inhibit BKB2R, or mediate immune response toward cells expressing this receptor, thus showing anti-cancer properties. This latter is another point of view that should be considered in the discussion.\nTaking together, these considerations suggest that advise against the flu vaccination is premature, while further studies that aim to clarify if the vaccine against influenza A viruses could elicit agonistic antibodies for BKB2R are needed and could have an impact on flu vaccination choice as well as on cancer treatments.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3530", "date": "22 Mar 2018", "name": "Veljko Veljkovic", "role": "Author Response", "response": "Response to Referee 2 Referee stated following: “These considerations are not solid enough to advise against the flu vaccination of cancer patients.” and “that advise against the flu vaccination is premature, while further studies that aim to clarify if the vaccine against influenza A viruses could elicit agonistic antibodies for BKB2R are needed and could have an impact on flu vaccination choice as well as on cancer treatments.” In this article authors not advise against flu vaccination but suggested “that until the role of the influenza vaccine in activation of BKB2R is clarified, vaccination of cancer patients against flu should be taken with some caution, and vaccines need to be monitored beyond the flu season”. Referee 1 gives the similar suggestion: “Most studies have limited follow-up, related to the seasonal nature of influenza vaccination. Prolonged monitoring of vaccinees therefore is warranted.” Antibodies directed against G-protein coupled receptors (GPCR) can act as receptor agonists or antagonists (see Dragun et al. Thromb Haemost. 2009;101:643). Taking into account (i) that activation of BKB2R pathway has cardioprotective effect and (ii) that influenza vaccine showed protection against cardiovascular disease, it was reasonable to hypothesize that antibodies elicited by influenza vaccine act as agonist of BKB2R. If these antibodies would act as antagonist of BKB2R than influenza vaccine should be harmful for CVD patients." } ] }, { "id": "32017", "date": "20 Mar 2018", "name": "Ger T Rijkers", "expertise": [ "Reviewer Expertise medical immunology (GTR)", "oncology (MW)", "influenza vaccination (both)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Paessler and Veljkovic on the potential link between bradykinin-2 receptor (BKB2R) and influenza virus is an interesting, as well as a provocative follow-up of previous work by this group published in 20141. In that paper, sequence homology between influenza virus and human proteins was investigated by the informational spectrum method (ISM analysis). BKB2R, although it didn’t get the highest amplitude or signal:noise values on the IS frequency, was studied in more detail because of its association with cardiovascular disease. It should be noted that conventional BLASTing of BKB2R with a range of influenza A strains (including A/Beijing/262/95 (H1N1), A/NewCaledonia/20/1999(H1N1)), shows very limited sequence homology. Thus far there is no direct evidence that influenza vaccination induces antibodies (agonistic or antagonistic) that would bind to BKB2R, but is interesting to speculate about possible consequences. Bradykinin, signaling via BKB2R (as well as BKB1R) is an important regulator molecule in inflammatory and vascular processes including angioedema, tissue permeability, vascular dilation, and smooth muscle contraction. Bradykinin also has been shown to promote proliferation and invasion of cancer cells, as indicated in the manuscript. Indeed, natural or experimental infection with influenza virus induces bradykinin secretion, which can be measured in nasal secretions during the first days after onset of symptoms (before specific antibodies are formed)2. In the above context, this would argue in favor of vaccination to prevent influenza infection. The immunogenicity and safety (including potentially negative effects) of influenza vaccination in (pediatric) patients with cancer have been published: NCT001121123, NCT009067504. In an accompanying editorial of latter study, the need for influenza vaccination of children with cancer is emphasized5. Most studies have limited follow-up, related to the seasonal nature of influenza vaccination. Prolonged monitoring of vaccinees therefore is warranted. Patients with solid tumors are treated with chemotherapy and/or checkpoint inhibition therapy. From that perspective, BKB2R agonists have been investigated as adjuvant therapy. While being effective in animal models6, thus far it is not being used in patients. It could be concluded that the intricate relation between bradykinin and influenza, also in the context of vaccination of patients with cancer required further study. Current clinical and immunological data however would not support a safety concern.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3531", "date": "22 Mar 2018", "name": "Veljko Veljkovic", "role": "Author Response", "response": "Response to Referee 1 The Referee’s statement: “In that paper, sequence homology between influenza virus and human proteins was investigated by the informational spectrum method (ISM analysis).” is wrong because ISM is the bioinformatics approach for analysis of the information encoded in the proteins. For this reason, results obtained by ISM and BLAST are incomparable. The same concerns the Referee’s remark that immunological cross-reactivity between influenza antigens and BKB2R is unlikely because of “very limited sequence homology”. The cross-reactivity between proteins with the common ISM frequency is experimentally proved, even in the absence of their sequence homology (Koma et al. Sci Rep 2018;8:1882). Referee’s conclusion that vaccine is beneficial for cancer patients because it prevents bradykinin secretion is correct, but it is not in contrast with conclusions in this article, which concern the different aspect the safety of influenza vaccine. It is important correctly assess positive and negative effects of vaccination for cancer patients taking into account a very low effectiveness of influenza vaccine. Referee pointed out some positive anticancer effects of activation of BKB2R in animal model. It is hard to believe that this will be tested in humans, taking into account numerous harmful effects of activation of BKB2R in cancer patients. Finally, Referee concluded “that the intricate relation between bradykinin and influenza, also in the context of vaccination of patients with cancer required further study.” and that: “Most studies have limited follow-up, related to the seasonal nature of influenza vaccination. Prolonged monitoring of vaccinees therefore is warranted.” It is exactly what we suggested in the conclusion of this article." } ] } ]	1	https://f1000research.com/articles/7-1
https://f1000research.com/articles/6-2012/v1	14 Nov 17	{ "type": "Research Note", "title": "The rise and fall of machine learning methods in biomedical research", "authors": [ "Hashem Koohy" ], "abstract": "In the era of explosion in biological data, machine learning techniques are becoming more popular in life sciences, including biology and medicine. This research note examines the rise and fall of the most commonly used machine learning techniques in life sciences over the past three decades.", "keywords": [ "machine learning", "linear regression", "support vector machine", "random forest", "deep neural network", "principal component", "t-SNE", "hierarchical clustering" ], "content": "Introduction\n\nOver the past three decades, biological data have grown dramatically in both size and complexity. The major contributors to the growth in size of computation biology data include, but not are not limited to, the ability of biologists to sequence complex genomes such as the human genome (1990–2003) (Lander et al., 2001), the advent of new high throughput sequencing techniques (around 2008) (Marx, 2013), and most recently the very rapid advancements in single cell technologies, introduced in 2009 (Wang & Navin, 2015).\n\nThe complexity of biological data has been growing even faster, and doesn’t seem to be linearly dependent on the size of data. Examples of complexity in the field of computational genomics include multiple diverse sources of technical noise, low signal to noise ratio, low numbers of biological replicates in comparative approaches, rare and usually hardly detectable mutations in non-coding regions and rare and barely identifiable cell types in complex heterogeneous systems such as the immune system and/or the brain.\n\nAt he intersection of mathematics, statistics and computer science is machine learning (ML), the de facto tool box in data science for deciphering the relationship between the input and output as well as detecting significant patterns within large, complex data sets. These quantitative approaches have been shown to be effective and are becoming increasingly popular in addressing challenges such as those outlined above. Highlights of their successful applications in functional genomics include, but are not limited to, learning and characterizing chromatin states by employing unsupervised approaches such as chromHMM (Ernst & Kellis, 2012), predicting sequence specificities of DNA- and RNA-binding proteins using convolutional neural networks such as DeepBind (Alipanahi et al., 2015), and employing a combination of supervised and unsupervised approach to determine the genetic and epigenetic contributors of antibody repertoire diversity (Bolland et al., 2016). Nowadays it is almost impossible to publish a study on single cell assays without using dimensionality reduction methods such as Principal Component Analysis or t-SNE.\n\nOne indirect measure of the success of these techniques in extracting scientific insights from biological data is to measure the popularity and usage of machine learning algorithms in life sciences research over time. I set out to quantify what fraction of published papers in the NCBI database mention a particular technique and how these numbers change over time.\n\n\nMethods\n\nFor this analysis, I used the R RISmed package (Kovalchik, 2015) to parse the publication data from NCBI. I examined publications in PubMed from 1990 to 2017 using a metric that measures the proportion of publications per year that mention the technique in the full text (Hits Per Year per Million articles published, or HPYM). The Popularity Rate (PR) of a technique was then defined as the difference between HPYMs in any two consecutive years. A positive PR shows an increase in popularity, whereas a negative PR reflects a decrease in popularity. I limited this note to 10 models listed in Table 1 which have been the most common or which showed a sharp change in popularity rate at a particular time. However, the R code is available with which any particular model during a specific period of time can be easily measured.\n\nThis table shows 10 machine learning techniques whose popularity in life sciences have been investigated in this study. Technical note: Supervised means that the model requires training data to learn its parameters. A supervised model is used to predict the future instances. An unsupervised model doesn’t require any training data and is used to detect patterns within a dataset. Dimensionality reduction models are used to project high-dimensional datasets into lower dimension space where new variables are more interpretable.\n\n\nResults\n\nThis analysis demonstrates that the overall popularity of machine learning methods in biomedical research has linearly increased since 1990 to 2017, but with two different slopes. From 1990 to 2000 the slope is 0.02, meaning that popularity increased only 2% per year. In 2000 (when sequencing big genomes became possible) the slope increased to 0.06, and since then it has remained constant. Perhaps surprisingly, a maximum of 1.2% of all papers published in PubMed in any calendar year have mentioned one of the machine learning methods investigated in this study (Figure 1A).\n\nA: Cumulative usage of all 10 machine-learning techniques. Two different linear regression models have been fitted to this data. The first one covers years from 1990 to 2000 and the second one that shows a triple increase in its slope, covers from 2000 to 2017. Y-axis shows number of hit per 100 publications. B: Trends of individual techniques, defined as per million hits in y-axis. C: The same as B but without Linear Regression and Principal Component Analysis.\n\nThe Linear Regression (LR) models have been the most dominant machine learning techniques in the life sciences over the past three decades (Figure 1B). It is interesting to see that their popularity rate has not been much effected by the rise of more sophisticated ML techniques such as ensemble-based approaches and/or Support Vector Machines and even with very recent and state of the art deep learning techniques. With a constant increase of 300 HPYM, and considering its higher intercept at 1990, the linear regression models is predicted to be one of the most popular techniques over the next few years.\n\nPerhaps a very surprising observation of this study is the rise and fall of Principle Component Analysis (PCA). PCA became very fashionable between 2000 and 2013. Since then it has been less used less, although it still is the second most popular tool (Figure 1B).\n\nIn early 2000s, unsupervised Hierarchical Clustering alongside newly introduced supervised techniques Support Vector Machines (SVMs) and Random Forests (RFs), showed a sharp rise in usage, which was mainly associated to microarray data analysis. Usage of hierarchical clustering plateaued shortly after its sharp popularity rise in 2000. SVMs kept their popularity longer, for almost a decade in fact, but subsequently dropped to an almost negligible popularity rate. RFs on the other hand, showed less popularity at the beginning of their arrival, but later on (after 2013) they were ranked the second highest in popularity after Deep Neural Networks (DNN) (Figures 1B and 1C).\n\nDuring the period between 1990–2017, neural networks have demonstrated considerable fluctuations in popularity. Known as Artificial Neural Networks (ANNs) in the early 1990’s after Linear Regression and PCA, they were the most commonly used techniques until early 2000, when they lost their popularity to MMs, HCs and SVMs and even later to RFs. However, since 2013, when they became known as Deep Neural Networks (DNNs), their usage has increased remarkably, so that they currently have the highest popularity rate (Figure 1B and 1C).\n\nThe dimensionality reduction technique t-distributed Stochastic Neighbour Embedding (t-SNE) published in 2008, has become quickly tailored to all sorts of single cell techniques. It is therefore not surprising to see that t-SNE usage has also been very rapidly growing over the past few years (Figure 1C).\n\n\nDiscussion\n\nI have illustrated the rise and fall of ML techniques in life sciences from 1990 to the present day. I chose this period because I believe this is the transition period for life scientists to join the big-data club. With the same R code used in this study to parse the publication data from NCBI, it would be possible to look at any period of time.\n\nIt was not very surprising to see LR models as the most commonly used model in the field, since:\n\na) LR models are one of the oldest ML methods that have been in use in almost any field,\n\nb) Parameters in LR models can be learned by using a training data with just a few data samples.\n\nc) A lot of other models can be placed under this umbrella, for instance by first applying a transformation function.\n\nIt was, however, surprising to see the sharp rise and fall of PCA. Perhaps a contributing factor to PCA being the most dominant dimensionality reduction method available in this period was its easy-to-use implementation in R. The question still remains as to why its popularity decreased from 2008 onwards. Perhaps the arrival of more versatile models such as RFs and SVMs which are very capable of handling high dimensionality and dealing with co-linearity in biological data eased the need to use PCA. Additionally, t-SNE as a tremendously growing dimensional reduction model in the field, is establishing itself as a strong competitor for PCA.\n\nANNs have been fairly popular since the 1990s until around 2004. Around that time more readily useable and less complex techniques became available, such as SVMs, RFs and MMs. However, with the huge investments of giant information companies such as Google leading to very impressive applications of ANNs (now known as DNNs) in various disciplines, their popularity has started to grow again. The sharp increase usage in popularity rate of DNNs over the past few years (Figure 1C) suggests that DNNs will take the PR lead again in the coming years.\n\nI appreciate that there are limitations to this study. For instance, for the majority of the comparative analyses of gene expression, researchers use a differential expression software and/or package, but cite only the package name and not the underlying statistical or ML technique used in the package. These cases have not been covered in this study. However, this study can be considered an approximation of the extent of machine learning techniques used in life sciences.\n\nIn a similar study (Jensen & Bateman, 2011), Jensen et al. investigated the rise and fall of only a few supervised machine learning techniques in life sciences. This study can be considered an update and extension of Jensen et al’s work, where the search for the mention of a particular technique was limited o the abstracts of papers in PubMed.\n\n\nData and software availability\n\nDataset 1: The text file contains the raw data underlying the results presented in this study, i.e. the number of publications in PubMed mentioning each machine learning technique from 1990–2017. These data is further normalized per million for downstream analysis. DOI, 10.5256/f1000research.13016.d184022 (Koohy, 2017).\n\nR code used to parse the publication data from NCBI is available at: https://github.com/hkoohy/Machine_Learning_in_Life_Sciences\n\nArchived source code as at the time of publication: http://doi.org/10.5281/zenodo.1039642 (hkoohy, 2017).\n\nLicense: GNU GENERAL PUBLIC LICENSE", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Human Immunology Unit MRC Core grant (MC_UU_12010).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nI am very grateful to David Sims, Edward Morrisey and Supat Thongjuea for critical reading of the manuscript and for their invaluable comments.\n\n\nReferences\n\nAlipanahi B, Delong A, Weirauch MT, et al.: Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning. Nat Biotechnol. 2015; 33(8): 831–838. PubMed Abstract \| Publisher Full Text\n\nBolland DJ, Koohy H, Wood AL, et al.: Two mutually exclusive local chromatin states drive efficient V(D)J recombination. Cell Rep. 2016; 15(11): 2475–2487. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErnst J, Kellis M: ChromHMM: automating chromatin-state discovery and characterization. Nat Methods. 2012; 9(3): 215–216. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nhkoohy: hkoohy/Machine_Learning_in_Life_Sciences: First release. (Version V1.0.0). Zenodo. 2017. Data Source\n\nJensen LJ, Bateman A: The rise and fall of supervised machine learning techniques. Bioinformatics. 2011; 27(24): 3331–3332. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoohy H: Dataset 1 in: The rise and fall of machine learning methods in biomedical research. F1000Research. 2017. Data Source\n\nKovalchik S: RISmed: download content from NCBI databases. R package version. 2015. Reference Source\n\nLander ES, Linton LM, Birren B, et al.: Initial sequencing and analysis of the human genome. Nature. 2001; 409(6822): 860–921. PubMed Abstract \| Publisher Full Text\n\nMarx V: Biology: The big challenges of big data. Nature. 2013; 498(7453): 255–260. PubMed Abstract \| Publisher Full Text\n\nWang Y, Navin NE: Advances and applications of single-cell sequencing technologies. Mol Cell. 2015; 58(4): 598–609. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "28048", "date": "27 Nov 2017", "name": "Alex Bateman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI should firstly point out that I was co-author on the 2011 editorial published in Bioinformatics titled, “The rise and fall of supervised machine learning techniques”1. Therefore I was momentarily surprised to be invited to review a paper with such a similar title. That editorial was only a page and a half long and only really scratched the surface of the interesting topic of the prevalence of use of machine learning in the biosciences. The author cites our 2011 paper and mentions that the current article can be considered an update of it. However, that is only mentioned in the very final paragraph of the paper. It would seem reasonable to me to make that one of the first things mentioned in the paper. Of course I am far from a neutral observer on this point.\nOverall I think that the article presents sound and interesting science and should be published within F1000Research. I think it provides a timely update to the 2011 editorial and expands it with some nice extra details. The article increases the number of ML methods investigated from 5 to 10. Most notably linear regression models are included which top the league table.\nI noticed an inconsistency in the data presented for ANNs in this new paper compared to the 2011 paper. Why is that? The numbers for ANN are considerably lower in this article. Is that because DNNs are split out from ANNs? Throughout the paper it says that ANNs have become known as DNNs. That is not correct. DNNs are a subtype of ANNs. So all DNNs are ANNs, but not all ANNs are DNNs. That needs correction throughout.\nThe following statement does not read well: \"The sharp increase usage in popularity rate of DNNs over the past few years (Figure 1C) suggests that DNNs will take the PR lead again in the coming years.\" After multiple readings I would presume that PR lead means it has the highest popularity rate. DNNs would have more than 300 more mentions per million papers per year. Firstly that sentence is very confusing to understand for a reader. For the first two readings I thought you were saying that DNNs would take the lead from LRMs, which would seem unlikely. On third reading I thought you meant that the slope of DNNs would overtake LRMs, but clearly it has already done that. I think you should rethink that sentence or take it out.\nMinor points:\nPage 2. At he intersection -> At the intersection Page 2. You mention that a surprising maximum of 1.2% of all paper mention one of the 10 ML techniques. Why is that surprising? Is it too low, too high? Please explain. Page 2. NCBI database is mentioned. NCBI has a lot of databases, please specify which one. Page 3. less used less -> used less\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3297", "date": "02 Jan 2018", "name": "Hashem Koohy", "role": "Author Response", "response": "I thank Dr. Alex Bateman for his time in evaluating the manuscript as well as for his very valuable comments.In fact, I was inspired by Alex’s commentary. I therefore apologize for not appropriately mentioning this earlier in the manuscript. I have made this change to the manuscript and I hope that is clear enough now.As Alex has suggested, I have made the distinction between ANNs and DNNs clear in corresponding paragraph and change the manuscript accordingly.I have also addressed the minor points accordingly. I hope the current version of manuscript meets Alex’s standards and consequently is clearer for the readers now." } ] }, { "id": "28432", "date": "06 Dec 2017", "name": "Konrad Förstner", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript \"The rise and fall of machine learning methods in biomedical research\" the author has generated a quantitative perspective on the usage of machine learning methods in the life sciences. For some of the methods a hypothesis about the underlying reason for an increased or decrease popularity are discussed. The code for performing the analysis is available on GitHub and - like the retrieved PubMed data - has been deposited at Zenodo.\nI have several major objections / question / suggestion for the author:\nI tried to reproduce the analysis using RStudio 1.1.383 with the deposited RStudio project but got the following error when executing the R chunks in the file Machine_Learning_Trends.Rmd: \"Error in library(informationRetrieval) : there is no package called ‘informationRetrieval’\" The file informationRetrieval.R is located in another subfolder and I guess this just needs proper referencing inside of the project. The author states that he has selected widely used machine learning methods used in life sciences. I would have expected Naive Bayes classifiers in the list of most popular methods. A simple PubMed search for '\"naive bayes classifier\" OR \"naive bayesian classifier' return twice as many hits as for \"deep neural networks\" (but over a longer time span):\nhttps://www.ncbi.nlm.nih.gov/pubmed/?term=%22naive+bayes+classifier%22+OR+%22naive+bayesian+classifier%2 https://www.ncbi.nlm.nih.gov/pubmed?term=%22deep+neural+networks%22\n\nSimilar issue for logistic regression: The analysis in the provided file Machine_Learning_Trends.Rmd actually contains the counting of publications containing logistic regression that shows a large (206,619 at the time of writing) and growing number of this but this method has not been discussed in the manuscript and is not displayed in the plots.\nhttps://www.ncbi.nlm.nih.gov/pubmed?term=%22logistic%20regression%22\n\nThe counting of hits for deep neural networks (DNN) is not done properly. Looking at the code to count the number of hits of different search terms shows that the author use \"artificial neural networks\" and \"deep neural networks\" and \"deep learning\" as search term for DNN (see code selection at the bottom of this section). I think using the search term \"artificial neural network\" for both ANN and DNN is not sound and changes the story of DNN (a special form ANN) significantly. Either DNN is treated as subset of ANN and only ANN are plotted or DNN and ANN are treated separately and the search term \"artificial neural network\" is not used for DNN. Furthermore the search term \"deep learning\" results in numerous unrelated hits before 2010 (e.g. PMID: 8936230, 9165817, 9487168, 10463930). https://www.ncbi.nlm.nih.gov/pubmed/?term=%22deep+learning%22 (then click on the \"Result by year\" histogram). The authors tries to explain the underlying reasons for the gain or loss of certain ML methods. In Figure 1 one of the publications of the human genome is placed in the year 2000 without any citation. The human draft genome was published in 2001 (International Human Genome Sequencing Consortium, Nature 409, 860–921, 2001, https://doi.org/10.1038/35057062) and it would be interesting to see what the author is referring to. The Popularity Rate (PR) introduced here is not plotted anywhere directly but is the slope of edges between the data points of two consecutive years. The author should consider visualizing this measurement of change as well. The curve plotted in Fig 1 A is nearly reassembled by the LRM curve in Fig 1 B. Is the observation in Fig 1 A maybe only an observation of the dominating LRM method? I do not understand why Fig 1 A can actually look nearly exactly like the LRM curve considering the other methods e.g. the PCA curve.\n\nCode selection regarding ANN and DNN ``` ANN_hits\n\n<- get_normliazed_number_of_hits(years = YEARs, query=\"artificial neural network[tw]\", db=\"pubmed\", normalization_value=1000000)\nNN_term <- \"(artificial neural networks[tw] OR deep neural networks[tw] or deep learning[tw])\" DNN_hits <- get_normliazed_number_of_hits(years=YEARs, query=NN_term, db=\"pubmed\", normalization_value=1000000) ```\n\nMinor issues:\nFigure 1\nStyle: The different lines are hard to distinguish by color only - maybe consider an additional discriminator (e.g. dashed lines for a subset); Next to Fig 1 C is a lot of white space. Placing the t-SNE subplot to a different location (e.g. the middle of Fig 1 C) would make it possible to use this space more efficiently. Maybe think rearranging the whole figure. Figure 1 C is a subplot of figure 1 B like the t-SNE plot is a subplot of Figure 1 C\n\n\"de facto\" should be written in in italic font The link to RISmed should use the link indicated at the page itself that says \"Please use the canonical form https://CRAN.R-project.org/package=RISmed to link to this page.\" For Linear Regression Model sometimes \"LRM\" and sometime \"LR\" is used in the manuscript In order to understand which biological approaches / questions that are influencing the usage of different ML method the association of those methods with certain MeSH term would be interesting. Either as part of this manuscript or a future one.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3298", "date": "02 Jan 2018", "name": "Hashem Koohy", "role": "Author Response", "response": "I thank Dr Konrad Forstner for his time in evaluating the manuscript and for his very detailed comments/suggestions that I believe will immensely enhance the quality of the manuscript. In the following I address the issues raised by Konrad. In the manuscript \"The rise and fall of machine learning methods in biomedical research\" the author has generated a quantitative perspective on the usage of machine learning methods in the life sciences. For some of the methods a hypothesis about the underlying reason for an increased or decrease popularity are discussed. The code for performing the analysis is available on GitHub and - like the retrieved PubMed data - has been deposited at Zenodo.I have several major objections / question / suggestion for the author: I tried to reproduce the analysis using RStudio 1.1.383 with the deposited RStudio project but got the following error when executing the R chunks in the file Machine_Learning_Trends.Rmd: \"Error in library(informationRetrieval) : there is no package called ‘informationRetrieval’\" The file informationRetrieval.R is located in another subfolder and I guess this just needs proper referencing inside of the project. I have changed the package structure and added a cell on top of the rdm file so that it should easily find the *.r codes and source them. The author states that he has selected widely used machine learning methods used in life sciences. I would have expected Naive Bayes classifiers in the list of most popular methods. A simple PubMed search for '\"naive bayes classifier\" OR \"naive bayesian classifier' return twice as many hits as for \"deep neural networks\" (but over a longer time span): https://www.ncbi.nlm.nih.gov/pubmed/?term=%22naive+bayes+classifier%22+OR+%22naive+bayesian+classifier%2 https://www.ncbi.nlm.nih.gov/pubmed?term=%22deep+neural+networks%22 In my very initial analysis I had naïve bayes classifier, but to my surprise after normalization it was overshadowed by other highly used techniques. I therefore took it out.Now, for the completion, I have added it again. Similar issue for logistic regression: The analysis in the provided file Machine_Learning_Trends.Rmd actually contains the counting of publications containing logistic regression that shows a large (206,619 at the time of writing) and growing number of this but this method has not been discussed in the manuscript and is not displayed in the plots. Similarly, I had logistic regression models in my initial analysis. And for the same reason I took it out from the final submission and left to the reader to test if they wish. It has been added again. https://www.ncbi.nlm.nih.gov/pubmed?term=%22logistic%20regression%22 The counting of hits for deep neural networks (DNN) is not done properly. Looking at the code to count the number of hits of different search terms shows that the author use \"artificial neural networks\" and \"deep neural networks\" and \"deep learning\" as search term for DNN (see code selection at the bottom of this section). I think using the search term \"artificial neural network\" for both ANN and DNN is not sound and changes the story of DNN (a special form ANN) significantly. Either DNN is treated as subset of ANN and only ANN are plotted or DNN and ANN are treated separately and the search term \"artificial neural network\" is not used for DNN. Furthermore the search term \"deep learning\" results in numerous unrelated hits before 2010 (e.g. PMID: 8936230, 9165817, 9487168, 10463930). https://www.ncbi.nlm.nih.gov/pubmed/?term=%22deep+learning%22 (then click on the \"Result by year\" histogram). I really apologize for this. I have corrected in the code, and changed the manuscript accordingly. The authors tries to explain the underlying reasons for the gain or loss of certain ML methods. In Figure 1 one of the publications of the human genome is placed in the year 2000 without any citation. The human draft genome was published in 2001 (International Human Genome Sequencing Consortium, Nature 409, 860–921, 2001, https://doi.org/10.1038/35057062) and it would be interesting to see what the author is referring to. I apologize again for the confusion. I was in fact referring 2001 IHGSC paper, as I had cited in the manuscript. I have changed the figure to make it clear. The Popularity Rate (PR) introduced here is not plotted anywhere directly but is the slope of edges between the data points of two consecutive years. The author should consider visualizing this measurement of change as well. Very valid point. I have restructured the manuscript and the figures. Now, I have a separate figure for this. The curve plotted in Fig 1 A is nearly reassembled by the LRM curve in Fig 1 B. Is the observation in Fig 1 A maybe only an observation of the dominating LRM method? I do not understand why Fig 1 A can actually look nearly exactly like the LRM curve considering the other methods e.g. the PCA curve. Great observation. Both figures are very similar, though with different slopes and intercepts. In order to check if the cumulative figure is dominant by LRM, in a separate task, I filtered out LRM and made the cumulative figure. Although in both full-model and filtered-model we can see two different slopes (from 1990 to 2000, from 2001 ot 2017), not surprisingly, the full model fits better. I think what happens is that around the time that PCA starts declining, we have an almost exponential increase from other models such as RF, SVM and later on from DNN. These collectively delute effect of PCA decline.Code selection regarding ANN and DNN```ANN_hits <- get_normliazed_number_of_hits(years = YEARs, query=\"artificial neural network[tw]\", db=\"pubmed\", normalization_value=1000000)NN_term <- \"(artificial neural networks[tw] OR deep neural networks[tw] or deep learning[tw])\"DNN_hits <- get_normliazed_number_of_hits(years=YEARs, query=NN_term, db=\"pubmed\", normalization_value=1000000)```Minor issues: Figure 1 Style: The different lines are hard to distinguish by color only - maybe consider an additional discriminator (e.g. dashed lines for a subset); Next to Fig 1 C is a lot of white space. Placing the t-SNE subplot to a different location (e.g. the middle of Fig 1 C) would make it possible to use this space more efficiently. Maybe think rearranging the whole figure. Figure 1 C is a subplot of figure 1 B like the t-SNE plot is a subplot of Figure 1 C As suggested, I have restructure the manuscript and the figures. The manuscript now has three main figures which are hopefully more clearer than the previous version. \"de facto\" should be written in in italic font Corrected. The link to RISmed should use the link indicated at the page itself that says \"Please use the canonical form https://CRAN.R-project.org/package=RISmed to link to this page.\" For Linear Regression Model sometimes \"LRM\" and sometime \"LR\" is used in the manuscript I have corrected for this. In order to understand which biological approaches / questions that are influencing the usage of different ML method the association of those methods with certain MeSH term would be interesting. Either as part of this manuscript or a future one. This is a very interesting point. Though as suggested is beyond this manuscript." } ] } ]	1	https://f1000research.com/articles/6-2012
https://f1000research.com/articles/6-2188/v1	29 Dec 17	{ "type": "Review", "title": "Infective endocarditis today", "authors": [ "Donato D'Agostino" ], "abstract": "Endocarditis is an infectious disease caused by numerous microorganisms, many of which habitually colonize the oral cavity and skin. Various bacteria and bacterial factors influence the duration of bacteremia and the possible colonization of cardiac valves. If not recognized and treated early, the disease may have serious consequences until death: despite the possibility of setting up targeted antibiotic therapy, it may occur in 30% of cases. This event is related to sepsis that develops in these patients and can result in cellular functional alterations in many districts, resulting in multi-organ failure (MOF) status. This paper is aimed to present an overview of this condition, based on the author’s own clinical experience and literature review.", "keywords": [ "Infective Endocarditis", "risk factors", "prophylaxis", "prophylactic therapy", "bacteraemia", "oral surgery", "oral hygiene", "Staphylococcus aureus." ], "content": "Introduction\n\nFrom an epidemiological point of view, the incidence of Infective Endocarditis (IE) varies considerably, depending on the socio-economic conditions of the various countries, with significantly lower rates in industrialized countries (USA 1.6–6 cases per 100,000 people per year). In Italy, according to recent surveys, the rate of incidence is between 1.7 and 6.2 per 100,000 inhabitants, with points of 3.6/100,000 inhabitants in the city of Turin and 4–5% in Veneto and Friuli Venezia Giulia Giulia. Most studies show that the male is most affected, with an M/F ratio of about 3–4: 1, probably due to the higher prevalence of bicuspid aorta in the male. Another predisposing factor is age. All age groups may be potentially affected but with different clinical picture between young immunosupressed subjects and older than 65 years have an incidence of about 9 times that of subjects with lower age1–4.\n\nIn the etiopathogenesis of the disease, some factors are linked to both the host and the responsible microorganism interacting with each other, which are: 1) the presence of predisposing conditions, 2) the presence of conditions capable of producing bacteriemia and 3) that the microorganisms involved are characterized by the ability to adhere and proliferate on the cardiac endothelium. In this context, mainly with regard to the predisposing conditions, mainly valvular diseases such as insufficiency and stenosis of the aortic and mitral valve (endocarditis site in 50% of cases) and the presence of valvular prostheses (12–17% of cases). Other predisposing conditions are congenital heart disease, such as interventional septal defects, Botallo's arterial channel persistence (4–18% of cases) and the use of injecting drugs (up to 30% in some cases). Causes of bacteremia can also be infectious processes in anatomic sites other than cardiovascular, as well as instrumental procedures such as bronchoscopy, colonoscopy, gastroscopy, cystoscopy, urethral and bladder catheterisation, urethrotomy, prostatectomy, hemodialysis, uterine endoscopy, and also abortion and/or delivery.\n\nIn the oral cavity, as well as the orotracheal intubation maneuvers, a source of bacteria that can give rise to endocarditis can be caused by dental infections and odontostomatologic maneuvers of treatment and prophylaxis or even poor oral hygiene, resulting in the onset of infectious diseases in periodontal tissues1–4.\n\n\nPathophysiology and clinical features\n\nThe main host factors involved in the pathogenesis of infective endocarditis are the valvular surfaces, leukocytes and platelets. The thrombus is formed on the valve surfaces and along the sutures in the valve prostheses. This thrombus is partly composed of platelet aggregates by bacterial induction and at the same time releases microbicidal proteins (contained in the d-granules), thus having a dual role in facilitating and limiting the progression of the disease. Leukocytes, via cytokines and complement, are responsible for immunopathological manifestations of infectious endocarditis, represented by hypergammaglobulinemia, both antigen-specific and linked to polyclonal activation of B cells (which can block the opsonizing IgG response, accelerate the microvascular damage or stimulation of phagocytosis), vasculitis caused by immune circulating complexes and reduction of complement for its consumption, as well as clinical syndromes such as deposition of circulating immune complex and complement, Osler nodules, etc.5.\n\nThe diagnosis of infective endocarditis is based on characteristic anatomopathological criteria such as the echocardiographic demonstration of pathological lesions (presence of vegetations or intracardiac abscesses confirmed by histological studies showing active endocarditis) and responsible microorganisms (proven presence by cultures or histological studies of in situ vegetation, or embolized vegetation or intracardiac abscess)6.\n\nThe experienced clinician can be oriented to the diagnosis by clinical and anamnestic elements. Some of these can lead to well-founded suspicion, as clinical features such as high fever (> 38C°), general condition (asthenia, myalgia), recent onset of cardiac murmur and signs of systemic embolization. Moreover, information can be derived from the patient's history about the execution of recent endoscopic instrumental maneuvers (cystoscopy, gastroscopy, colonoscopy, hysteroscopy) or invasive maneuvers (catheterization), especially in the elderly, recent surgery (especially valvular prosthesis), recent dental interventions (dental avulsion, periodontal surgery, endodontic treatment, dental implant surgery). It is also very relevant to the patient anamnesis if they are, or have been, a drug addict with use of injectable drugs5,7.\n\nAmong the less significant indices, the presence of a few days of febrile (around 37°C) should be mentioned, albeit associated with good systemic conditions. In addition, considering that bacterial endocarditis, if not diagnosed and treated quickly, may be burdened by poor prognosis, it is important to point out that, regardless of clinical/anamnestic suspicion, it should be considered and investigated (by serial emoculture and trans-esophageal echocardiogram), and above all, it should be excluded when the cause of the fever is not quickly detected. In addition, if a septic spot is detected elsewhere, it is advisable, to investigate the presence or absence of endocarditis, as this may also manifest itself as a complication.\n\nThe interval between the onset of infection and the onset of early symptoms is generally short (two weeks in most cases). However, it can be months, as in cases of infections caused by uncommon germs, or in the case of endocarditis on valve prostheses.\n\nGeneral symptomatology usually is hyperpyretis (febrile in most cases, high fever during septic shock), anorexia, asthenia, myalgia. Other symptoms may be related to so-called \"local\" effects: valve lesion/destruction with severe hemodynamic consequences or systemic embolization phenomena8.\n\nThe main clinical signs are: the classic Osler nodules, small nodules purple in color at the fingertips, pathognomic of endocarditis and to be attributed to probably embolic phenomena, cardiac murmurs (which may be indicative of new appearance or aggravation of previous heart disease), petechiae, consisting of small \"haemorrhagic\" spots often located at the conjunctiva of the oral cavity mucosa (related to microembolic phenomena and increased capillary permeability), and also present at the retinal level (Roth stains, red spots with a light center coming from hemorrhagic and inflammatory phenomena), as well as clubbing of the fingers.\n\nHeart complications are related to: 1) dysfunction of the native valve or valve prosthesis, resulting in acute valve failure resulting in a clinical picture of severe hemodynamic impairment, associated with 2) heart failure (found more frequently in aortic valve infection) and burdened by an unfavorable prognosis; or 3) myocardial infarction due to cardiac abscesses (in 30% of native endocarditis and 50% of prosthesis), often complicated by atrio-ventricular block5,8.\n\nThe prognosis of infective endocarditis, in various clinical forms, is progressively worsening in the following order: acute, staphylococcal, on valvular prosthesis, mycotic, refractory to medical treatment5,8\n\n\nMicrobiological aspects\n\nAny microorganism can potentially cause infective endocarditis, but infection is more commonly determined by specific microorganisms, particularly streptococci and staphylococci that show tropism for valve structures. They have two properties: 1) frequent association with bacteriemia, transient or persistent, and 2) ability to adhere to the surface of valves or thrombi due to the production of certain substances. Recent scientific works, however, indicates that oral microorganisms (especially Streptococcus spp.) are seldom found in bacterial endocarditis, while others such as S. aureus and enterococci are more frequently involved. The main causal factors of bacteremia, which usually originate from the skin or oral cavity, are dental brushing or gold-dental, genitourinary and gastrointestinal procedures, poor oral hygiene and dental sepsis, but also cellulitis and soft tissue infections, injecting drugs, intravascular catheterization, cardiac surgery (including pace-maker placement)9. Responsible microorganisms are constituted, in addition to bacteria of the genus Streptococcus spp. and Staphilococcus spp., also from rickettsia, chlamydia, fungi, C. burneti. Most infectious endocarditis on native valves or on cardiac valve prostheses are caused by streptococci. Generally, the responsible strain belongs to the group of virulent streptococci (S. sanguinis, oralis, salivarius, mutans) normally present in oropharynx and gastrointestinal tract. These are non-virulent pathogenic bacteria, which can cause transient bacteriemia and adherence to the endocardial system, but are extremely widespread in the population and in the case of very frequent oral pathologies such as caries10.\n\nIn the elderly population, in the lungs, the presence of Streptococcus bovis, a microorganism associated with polyps and colon carcinoma, or enterococci (Group D streptococci as Enterococcus faecalis) is commonly found in the gastrointestinal tract and can cause subacute infections after gastrointestinal clinical procedures. These bacteria are often selected by the use of broad spectrum antibiotics and are also found in the genitourinary tract and in young women, as a result of obstetric procedures, subacute infections may result. It is much less common to find, as responsible of IE, other streptococci such as S. pneumoniae. In the various literature available, Staphylococci are responsible for one third of endocarditis on naive valves and half of endocarditis on prosthetic valves.\n\nStaphilococcus aureus, a highly virulent positive coagulase bacterium, causes florid vegetation of the valve flaps causing their destruction, peripheral abscesses, severe dissected and metastatic infections, and is the main cause of acute endocarditis and endocarditis in drug addicts, while coagulasi negative S. epidermidis is the main cause of early endocarditis on valve prostheses. This microorganism is present on the skin and can contaminate catheter and vascular access, used in the post-operative course in Cardiac Surgery Units.\n\nGram-negative bacteria are rarely involved as IE responsible of, but are increasingly recognized as a cause of endocarditis in drug addicts and cardiac valve prostheses, Gram-negative Bacillus species belonging to the genera Pseudomonas, Brucella, N. gonorreae and those that grow slowly of the so-called HACEK group (Hemophilus, Actinobacillus, Cardiobacterium, Eikenella, Kingella).\n\nStatistically, 5–20% of endocarditis has negative emoculture, usually due to partial antibiotic treatments, fungal endocarditis, or to infections sustained by mutated streptococci, C. burnetti, Clami-dia, Bartonella, Legionella11.\n\nIn view of the variety of microbial species involved in the genesis of IE, it is necessary to set up targeted antibiotic therapy, which is only possible if properly cultivated. This selective choice of the drug obviously aims to avoid ineffective antibiotic therapies. Indeed, as noted above, endocarditis can (though rarely) be sustained by viruses or fungi on which antibiotics do not perform any action. In fact, in the case of the Candida and Aspergillus genera, that are responsible for a fair amount of endocarditis on native and prosthetic endocarditis and endocarditis in drug addicts. They determine the formation of large vegetations, phenomena of embolization and peripheral abscesses, whose therapy is difficult and often requires surgical treatment9,11.\n\n\nProphylaxis\n\nDespite the possibility of performing microbiological and instrumental elective investigations and the wide availability of drugs capable of resolving pathological conditions, IE is still associated with a high mortality rate. Therefore, in addition to diagnosis and early treatment of the disease, prevention can be implemented through the application of correct hygienic-behavioral norms and by pharmacological prophylaxis protocols. In this respect, we note that, in the literature and the evidence of clinical practice, when a history of probable cause is found among the most frequent statistical-epidemiological forms, besides the forms correlated to toxic-dependencies, IE are of primary relevance related to dental procedures in general and/or oral surgery, and they have a much higher incidence than the other types of instrumental maneuvers, and endoscopic procedures in general.\n\nAmong the causes of IE, for which prevention and control are more important, one can also include: care in insertion and management of vascular access, maintenance of skin hygiene in cardiac surgery patients, and immediate removal of infected catheters, in order to prevent infectious complications, septic embolisms from \"biofilm\". However, as mentioned above, oral and dental hygiene plays a major role in IE prevention.\n\nIn this regard too, in patients at risk of developing endocarditis and who are undergoing oral surgery and any dental procedure, prophylactic antibiotic therapy is desirable12.\n\nRelations between oral hygiene conditions, dental therapeutic procedures and infective endocarditis have been studied by several cardiological scientific societies that have put forward many guidelines for pharmacological prophylaxis in subjects at risk. In 1955, the AHA (American Heart Association) recommended the antibiotic prophylaxis of bacterial endocarditis during dental procedures. Since then, despite the lack of scientific evidence of its effective preventive efficacy, antibiotic-prophylactic prescription has become common practice in all dental procedures considered at risk of infectious sequelae13–15.\n\nThe list of subjects at risk has undergone major changes in the light of the evolution of scientific results. In 2007 guidelines for antibiotic prophylaxis were been revised and, according to the current guidelines proposed by the AHA and the British Society for Antimicrobial Chemotherapy (BSAC) and also implemented by AIFA (Italian Drugs Agency), there are four conditions that make antibiotic prophylaxis necessary before a dental procedure of any kind : 1) the presence of valve prostheses, 2) previous endocarditis, 3) previous pulmonary or systemic shunt interventions, and 4) cardiac transplant patients developing cardiac valvulopathy16–21. This proposal was confirmed by the European Society of Cardiology (ESC) in 2009, and later in 2015, producing a summary card for General Practice. Compared to the previous guidelines, dating back to 1997, the number of heart disease considered at risk of endocarditis was reduced, once divided into high, medium and low risk. Therefore, at present, the aforementioned cardiac conditions simply come under the only category of \"heart disease with an endocarditis risk\". A study was conducted to assess the doubts raised by a 2004 Cochrane review on the real effectiveness of antibiotic prophylaxis and this research would show that to prevent a few cases of infectious IE (2.7% of the population studied) the balance of the risk/benefit ratio should definitely be on the risk20–22.\n\nIt has also been underlined that there is a need for high levels of oral hygiene in patients undergoing cardiac surgery to be maintained over time with regular oral hygiene sessions, during which a further precaution, and not only in subjects known to be at risk, is provided by the use of oral antimicrobials (liquid collutants or antibiotic gels) prior to any oral cavity manipulation that will help to reduce potentially pathogenic microbial levels.\n\nHowever, it should be noted that some studies do not recognize the usefulness of antibiotic-prophylaxis in those at risk of having to undergo dental treatments of any kind, such as the aforementioned metanalysis conducted by the Cochrane Group.\n\nOver the years numerous antibiotic-prophylaxis protocols have been proposed for dental patients, including numerous antibiotics (Penicillin, Vancomycin, Ampicillin, Gentamicin, Azithromycin) and cephalosporins (Cefalexin, Cefadroxil), but currently the most widely used antibiotic is Amoxicillin (for subjects with history of allergy to penicillin, alternative molecules may be Clindamycin or Claritromycin) to be administered orally 60 minutes before the onset of surgery, with possible extension to 24 hours in particular cases. In the case of renal failure, the single dose administration of the drug does not need to be modified.\n\n\nConclusions\n\nInfective endocarditis (IE) is a cardiac pathology of bacterial, mycotic or more rarely viral origin, developing on the surfaces of the endocardium or heart valves. Predisposing conditions are congenital malformations of the heart or valvular acquired alterations, as well as the presence of a valvular prosthesis5. Still now IE causes death in 20–30% of patients.\n\nThe microorganisms involved in the etiology and pathogenesis of the damage of such infection determine the formation of the endocardic vegetations typical of this condition. Such lesions can be located on the valvular or the parietal endocardium and sometimes on the endothelium of a great artery. Despite the elevated standards of instrumental investigations and therapeutic protocols, IE represents a disease of wide interest, scientifically and socially, due to its high rate of incidence, morbility and mortality, usually due to MOF23.\n\nIt is therefore essential to evaluate laboratory parameters, both traditional and innovative, to prevent these consequences through targeted antibiotic therapy24.\n\nThe opportunity to implement antibiotic-prophylaxis in dental patients still remains a \"vexata quaestio\" and the analysis of literature demonstrates how the available data are somewhat controversial. Its application in everyday practice is based on other than efficacy tests: simply empirical or \"traditional\" tests19–21.\n\nCommon dental, professional and non-dental procedures, as well as many oral surgery maneuvers, can result in bacteria that are sustained by particularly aggressive and resistant bacteria. It should also not be forgotten that, following such maneuvers, it is possible to cause the penetration into the bloodstream of other microorganisms commonly present in the oral cavity (viruses and fungi), which are particularly difficult to identify and treat. Considerable progress has been made in the prevention of infectious diseases and cross-contamination in the urological, gynecological, gastrointestinal and stomatological instrumental procedures. However, in the latter procedure in particuler, from the ablation of the tartar to the oral cancer surgery, this still represents an important risk factor for the development of IE in predisposed patients. However, the actual responsibility of oral bacterial flora in the determinism of distant infections is still unclear, which is attempted to prevent antibiotic-prophylaxis during dental procedures9,10,12,20. This is the reason for the importance of prophylaxis in dental risk patients, which can be obtained, on the one hand, with careful patient evaluation and rational use of antibiotics and, on the other hand, with scrupulous and professional oral hygiene, associated with the main classical foundament of clinical practice, well known from antiquity: the empathy with patients25.\n\nIn conclusion, we can state that it is very important therefore, in order to limit the risks to the health of the patient and to avoid possible legal consequences of medical treatment, the collection of a thorough history of oral and/or dental history and continuous updating of the international guidelines, considering increased risk of cardiac complications in diabetic patients26. In 20% of IE cases apparently there is no predisposing factor, however, the role of molecules contained in cigarette smoke and liquids used for electronic cigarettes also cannot be excluded27.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nD’Agostino D, Bottalico L, Santacroce L: Infective endocarditis: what is changed in epidemiology and prophylaxis. Acta Medica Mediterranea. 2012; 28: 311–9. Reference Source\n\nMask AG Jr: Medical management of the patient with cardiovascular disease. Periodontol 2000. 2000; 23(1): 136–41. PubMed Abstract \| Publisher Full Text\n\nMangini F, Santacroce L, Bottalico L: Periodontitis and systemic diseases. Clin Ter. 2006; 157(6): 541–8.\n\nDuval X, Alla F, Hoen B, et al.: Estimated risk of endocarditis in adults with predisposing cardiac conditions undergoing dental procedures with or without antibiotic prophylaxis. Clin Infect Dis. 2006; 42(12): e102–7. PubMed Abstract \| Publisher Full Text\n\nFriedlander AH, Marshall CE: Pathogenesis and prevention of native valve infective endocarditis in elderly dental patients. Drugs Aging. 1994; 4(4): 325–30. PubMed Abstract \| Publisher Full Text\n\nLessard E, Glick M, Ahmed S, et al.: The patient with a heart murmur: evaluation, assessment and dental considerations. J Am Dent Assoc. 2005; 136(3): 347–56; quiz 380–1. PubMed Abstract \| Publisher Full Text\n\nBottalico L, Tatullo M, Marrelli M, et al.: Lights and shadows of dental implants: focus on mucositis and perimplantitis and their biological markers. J Biol Regul Homeost Agents. 2016; 30(3): 859–861. PubMed Abstract\n\nWeinberger I, Rotenberg Z, Zacharovitch D, et al.: Native valve infective endocarditis in the 1970s versus the 1980s: underlying cardiac lesions and infecting organisms. Clin Cardiol. 1990; 13(2): 94–8. PubMed Abstract \| Publisher Full Text\n\nUntheroth WG: How important are dental procedures as a cause of infective endocarditis? Am J Cardiol. 1984; 54(7): 797–801. PubMed Abstract \| Publisher Full Text\n\nBottalico L, Valenzano A, Leone D, et al.: [The incidence of dental caries during childhood. A clinical and epidemiologic study in Matera (Southern Italy)]. Clin Ter. 2007; 158(5): 409–19. PubMed Abstract\n\nBayliss R, Clarke C, Oakley C, et al.: The teeth and infective endocarditis. Br Heart J. 1983; 50(6): 506–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nD’agostino D, Losacco T, Santacroce L: Clinical management of the infective endocarditis today. Acta Med Mediterr. 2012; 28: 321–29. Reference Source\n\nVerhaaren H, Claeys G, Verschraegen G, et al.: Endocarditis from a dental focus. Importance of oral hygiene in valvar heart disease. Int J Cardiol. 1989; 23(3): 343–7. PubMed Abstract \| Publisher Full Text\n\nLittner MM, Kaffe I, Tamse A, et al.: New concept in chemoprophylaxis of bacterial endocarditis resulting from dental treatment. Oral Surg Oral Med Oral Pathol. 1986; 61(4): 338–42. PubMed Abstract \| Publisher Full Text\n\nHupp JR: Changing methods of preventing infective endocarditis following dental procedures: 1943 to 1993. J Oral Maxillofac Surg. 1993; 51(6): 616–23. PubMed Abstract \| Publisher Full Text\n\nO’Sullivan J, Anderson J, Bain H: Infective endocarditis in children following dental extraction and appropriate antibiotic prophylaxis. Br Dent J. 1996; 181(2): 64–5. PubMed Abstract \| Publisher Full Text\n\nLeport C, Horstkotte D, Burckhardt D: Antibiotic prophylaxis for infective endocarditis from an international group of experts towards a European consensus. Group of Experts of the International Society for Chemotherapy. Eur Heart J. 1995; 16(suppl B): 126–31. PubMed Abstract\n\nAl-Karaawi ZM, Lucas VS, Gelbier M, et al.: Dental procedures in children with severe congenital heart disease: a theoretical analysis of prophylaxis and non-prophylaxis procedures. Heart. 2001; 85(1): 66–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSantinga JT, Fekety RF Jr, Bottomley WK, et al.: Antibiotic prophylaxis for endocarditis in patients with a prosthetic heart valve. J Am Dent Assoc. 1976; 93(5): 1001–5. PubMed Abstract \| Publisher Full Text\n\nWilson W, Taubert KA, Gewitz M, et al.: Prevention of infective endocarditis: guidelines from the American Heart Association: a guideline from the American Heart Association Rheumatic Fever, Endocarditis and Kawasaki Disease Committee, Council on Cardiovascular Disease in the Young, and the Council on Clinical Cardiology, Council on Cardiovascular Surgery and Anesthesia, and the Quality of Care and Outcomes Research Interdisciplinary Working Group. J Am Dent Assoc. 2007; 138(6): 739–45, 747–60. PubMed Abstract\n\nGould FK, Elliott TS, Foweraker J, et al.: Guidelines for the prevention of endocarditis: report of the Working Party of the British Society for Antimicrobial Chemotherapy. J Antimicrob Chemother. 2006; 57(6): 1035–42. PubMed Abstract \| Publisher Full Text\n\nOliver R, Roberts GJ, Hooper L: Penicillins for the prophylaxis of bacterial endocarditis in dentistry. Cochrane Database Syst Rev. 2004; (2): CD003813. PubMed Abstract \| Publisher Full Text\n\nTrentadue R, Fiore F, Massaro F, et al.: Induction of mitochondrial dysfunction and oxidative stress in human fibroblast cultures exposed to serum from septic patients. Life Sci. 2012; 91(7–8): 237–43. Erratum in: Life Sci. 2013 May 2; 92(14–16): 873. PubMed Abstract \| Publisher Full Text\n\nDi Serio F, Lovero R, D’agostino D, et al.: Evaluation of procalcitonin, vitamin d and c-reactive protein levels in septic patients with positive emocoltures. Our preliminary experience. Acta Medica Mediterranea. 2016; 32: 1911–4. Publisher Full Text\n\nSantacroce L, Bottalico L, Charitos IA: Greek Medicine Practice at Ancient Rome: The Physician Molecularist Asclepiades. Medicines (Basel). 2017; 4(4): pii: E92. PubMed Abstract \| Publisher Full Text\n\nSantacroce L, Carlaio RG, Bottalico L: Does it make sense that diabetes is reciprocally associated with periodontal disease? Endocr Metab Immune Disord Drug Targets. 2010; 10(1): 57–70. PubMed Abstract \| Publisher Full Text\n\nTatullo M, Gentile S, Paduano F, et al.: Crosstalk between oral and general health status in e-smokers. Medicine (Baltimore). 2016; 95(49): e5589. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30072", "date": "05 Feb 2018", "name": "Bernard Prendergast", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Is the topic of the review discussed comprehensively in the context of the current literature? No\n\nMost writers would accept that the current literature reflects work published over the past 10 years. The majority of citations in this article are significantly older than this and largely relate to the field of dentistry rather than clinical medicine. I do not feel that the article reflects current practice, nor does it explore current controversies in the field.\n\nThere is no mention of the Duke Criteria or the modified update for the diagnosis of infective endocarditis. Diagnosis is usually made without histological confirmation at the time of surgery, which is reflected in the Duke Major criteria. Most would agree that the pathological criteria of endocarditis are largely historical.\n\nIn 2015 new imaging modalities were introduced into the ESC guideline to aid diagnosis including PET/SPECT, CT and imaging for distal embolic events. The article makes no reference to any imaging outside of TOE. Both the AHA and ESC would also recommend TTE rather than TOE as the first-line test for suspected disease.\n\nIn terms of antibiotic prophylaxis, the author does highlight that this is a controversial area. However, it might be helpful to reference current UK guidelines that do not recommend any prophylaxis, which has been the case since 2008. Potentially as a result of this, UK endocarditis rates are now rising.\n\nThe article does not reference or highlight the shift in microbiology from Streptococcus to staphylococcus or why this might be.\n\nThere is no mention of newer percutaneous valves and the signal regarding potential higher endocarditis rates.\n\n2. Are all factual statements correct and adequately supported by citations? No\n\nThere are frequent factual statements made throughout the article that are not referenced. These need to be addressed prior to publication. At present the article reads like an opinion piece.\n\nFor example.\n\n“endocarditis site in 50% of cases” “12-15% of cases” “injecting drugs (up to 30% in some cases)” “oral microorganisms (especially Streptococcus app.) are seldom found in bacterial endocarditis” ‘Statistically 5-20% of endocarditis has negative emoculture”\n\n3. Is the review written in accessible language? Partly\n\nThere are a number of esoteric terms used in the article that are not commonly used and make it challenging to read:\n\nEmoculture Anatomopathological Odontostomatologic Asthenia Anamnesis Hyperpyretis\n\nThe structure of some sentences is confusing:\n\n“All age groups may be potentially affected but with different clinical picture between young immunosupressed subjects and older than 65 years have an incidence of about 9 times that of subjects with lower age”\n\n“In the elderly population, in the lungs, the presence of Streptococcus bovis, a microorganism associated with polyps and colon carcinoma, or enterococci (Group D streptococci as Enterococcus faecalis) is commonly found in the gastrointestinal tract and can cause subacute infections after gastrointestinal clinical procedures”\n\n“Despite the possibility of performing microbiological and instrumental elective investigations and the wide availability of drugs capable of resolving pathological conditions, IE is still associated with a high mortality rate”\n\n“It’s application in everyday practice is based on other than efficacy tests: simply empirical or traditional tests”\n\n“which is attempted to prevent antibiotic-prophylaxis during dental procedures”\n\n4. Are the conclusions drawn appropriate in the context of the current research literature? Partly\n\nNo mention of more current risk factors for endocarditis: dialysis, immunosuppression, implantable cardiac devices or indwelling venous catheters. Agreed that mortality remains high. However, might be nice to discuss why this could be?\n\nThe reference to MOF in the second paragraph of the conclusion is rather confusing. It has not been mentioned in the article, the acronym is not described and I am unsure that mortality in endocarditis is “usually” due to it.\n\nAgreed that antibiotic prophylaxis remains controversial.\n\nThe reference to diabetic patients at the end of the article comes out of the blue. Diabetes is a risk factor for endocarditis, but it is not highlighted anywhere else in the article. This needs to be introduced earlier.\n\nThe throw-away statement regarding cigarette smoke and electronic cigarettes is also confusing. It bears no relation to the article and seems an odd thing to conclude what is quite a traditional article on endocarditis.\n\nSummary\n\nAll factual statements need to be referenced, ideally with articles <10 years old to ensure they reflect the current literature.\n\nClean up some sentences and try to move away from esoteric terms to make the article easier to read.\n\nThe second and last paragraphs of the conclusion need to be tightened up. At present they do not bear any relation to the overall article.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Partly\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-2188
https://f1000research.com/articles/6-2182/v1	28 Dec 17	{ "type": "Case Report", "title": "Case Report: Treatment of systemic mastocytosis with sunitinib", "authors": [ "Gerhard J. Molderings", "Lawrence B. Afrin", "Hans-Jörg Hertfelder", "Stefan Brettner", "Lawrence B. Afrin", "Hans-Jörg Hertfelder", "Stefan Brettner" ], "abstract": "Mast cell activation disease typically presents as chronic multisystem polymorbidity of generally inflammatory ± allergic theme. Presently, treatment of the rare, cytoproliferative variant systemic mastocytosis employs empirically selected therapies to impede mast cell mediator production and action and, when necessary, inhibition of proliferation. Some tyrosine kinase inhibitors (TKIs) have been used successfully in uncommon cases of systemic mastocytosis not bearing that disease’s usual imatinib-resistant KITD816V mutation. Recently, sunitinib, a multi-targeted TKI, had been successful in a case of systemic mast cell activation syndrome. In addition, most allergy is principally a mast cell activation phenomenon, and sunitinib has been shown helpful in controlling a murine model of oral allergy syndrome. Here, we present the first use of sunitinib in systemic mastocytosis.", "keywords": [ "mast cell", "systemic mast cell activation disease", "systemic mastocytosis", "sunitinib", "tyrosine kinase inhibitors" ], "content": "Introduction\n\nSystemic mastocytosis (SM) represents the rare variant (prevalence in Europeans ranges between 0.3 and 13:100,000)reviewed in 1 of systemic mast cell activation disease (MCAD; Table S1). MCAD comprises a heterogeneous group of multifactorial, polygenic disorders characterized by aberrant release of variable subsets of mast cell (MC) mediators together with accumulation of either morphologically altered and immunohistochemically identifiable mutated MCs due to MC proliferation (SM and mast cell leukemia) or morphologically ordinary MCs due to decreased apoptosis (well-differentiated SM and systemic mast cell activation syndrome)for details, see 2.\n\nDue to its genetic and epigenetic roots, MCAD generally is regarded as incurable. Recent mutational studiesreviewed in 3 revealed an individual pattern of genetic and epigenetic alterations for each patient, which may affect the receptor expression, and their intracellular signal transduction pathways. Hence, mediator formation and release as well as inhibition of apoptosis, and/or increase in proliferation, are determined by individual genetic and epigenetic conditions requiring a highly personalized therapy for the disease. In formal studies in SM patients, although some tyrosine kinase inhibitors (TKIs) reduced MC burden, as reflected by histological normalization in bone marrow and improved laboratory surrogate markers (e.g., blood tryptase level), at best only partial improvement of mediator-related symptoms was achieved4–7. Distinction in pathways in the MC that promote MC proliferation vs. mediator production/release may explain why TKIs reduce MC burdens and MC-driven symptoms to different degrees4–7. However, in some case reports, TKIs have been significantly effective at relieving symptoms. Thus, in spite of a potential for serious adverse effects of these drugs, therapeutic trials may be justified in individual cases at various stages in their courses. Possibly due to the causative mutations in multiple genes leading to simultaneous activation of multiple intracellular pathways, multi-targeted TKIs such as midostaurin and sunitinib may be more effective than drugs which selectively downregulate only one intracellular pathway. Sunitinib has been shown to be helpful in a murine model of oral allergy syndrome, a disease likely rooted in aberrant MC activation8.\n\n\nCase presentation\n\nA 58-year-old man presented to our Interdisciplinary Multicenter Research Group for Systemic Mast Cell Diseases for therapy of his progressive aggressive systemic mastocytosis (ASM, Table S1 and Table S2), diagnosed according to the WHO criteria (Table S3), which remained significantly symptomatic despite the use of drugs administered to reduce MC activationreviewed in 9 (prednisone, rupatadine, ranitidine, ascorbic acid, ketotifen, montelukast, omalizumab) and drugs administered to reduce mediator-related symptoms (omeprazole, candesartan, risedronic acid, clonidine, cholestyramine, tranexamic acid, metamizole). A previous therapeutic trial with methotrexate was unhelpful. As typical in SM, his full history is complex, and symptoms reported, and key laboratory results found, are summarized in Table 1. The only minimal elevation in tryptase in this case of not just SM but in fact aggressive SM illustrates the heterogeneity of SM/ASM.\n\nSince recently sunitinib had been used successfully in a case of systemic mast cell activation syndrome10 (Table S1), we decided for an off-label trial with sunitinib. Sunitinib is a multi-targeted TKI (up to 313 potential kinase targets)reviewed in 9 which, in addition to KIT, also binds to PDGFR-α, PDGFR-β, VEGFR1, VEGFR2, VEGFR3, FLT3, CSF-1R, and RET, some of which are also expressed in MCs. The patient gave written informed consent to participate in the off-label therapeutic trial with sunitinib, which is approved to treat imatinib-resistant, largely KIT-mutation-driven gastrointestinal stromal tumor and other applications, but not yet systemic mastocytosisreviewed in 11. For such a therapeutic trial, ethical approval is not necessary in Germanya. There was no contra-indication for use of sunitinib in the patient, in particular no sign of abdominal aortic aneurysm. We now report the first use of sunitinib in systemic mastocytosis.\n\nIn a first attempt, the patient took 12.5 mg sunitinib once daily for 24 days. After just three days, the abnormal bleeding (e.g. intense gum bleeding) he had due to increased fibrinolysis, which is a typical symptom in MCAD12,13, ceased. The multiple subcutaneous fibrotic nodules that had developed all over his body during his many years of SM became tender and movable in the skin. Although no other symptoms were improved and sunitinib did not prevent flares of the disease, the patient felt better subjectively, in particular with less fatigue. However, in parallel the body hair became depigmented (white) and there was a decrease both in the number of thrombocytes and in the amount of total protein in blood, whereas uric acid in the blood increased inducing gout (Table 1). At that point treatment with sunitinib was discontinued.\n\nWhen the altered blood parameters had returned to the normal ranges 33 days later, the patient asked for a second treatment try. During that time, the positive effects of the first period of sunitinib use had vanished. This second treatment, again with 12.5 mg sunitinib once daily, lasted only 8 days, during which the same positive and negative effects occurred as during the first intake, so the second therapeutic trial had to be terminated, too. It is possible that an even lower dose of the drug (e.g., 6.25 mg, which would pose some (surmountable) challenges, since the lowest capsule dose available is 12.5 mg), might have provided equal benefit with less toxicity, especially in view of the many case reports now of imatinib-responsive SM showing remarkable sensitivity to doses of that drug far below what seems necessary in most other oncologic applications of the drug. Cyclic rather than continuous dosing of sunitinib was shown to be the optimal approach in the approved indications for that drug. It may be the same, albeit using a cycling pattern different than for oncologic/anti-neoplastic applications, might prove true with use of sunitinib in SM.\n\n\nConclusions\n\nIn conclusion, this ASM partially responded to sunitinib. However, in this patient toxicities outweighed limited benefits. A similar benefit-risk assessment of sunitinib was observed in a second patient with ASM (unpublished case; GJM). However, our findings do not rule out that therapeutic trials with sunitinib may be reasonable in other SM patients whose disease is refractory to other TKIs.\n\n\nConsent\n\nWritten informed consent for publication of his clinical details was obtained from the patient.\n\n\nNotes\n\na In Germany, individual therapeutic trials are not regulated by law. They are not notifiable, and there is no register for it. It is recognized that the freedom of medical treatment also includes the individual therapeutic attempt. The freedom of medical treatment has a legal basis in sub-constitutional laws and in regulations of the German medical association. In the end it is based on the freedom to pursue an occupation guaranteed by article 12 subparagraph 1 of the German constitution.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nTable S1. Classification of mastocytosis.\n\nClick here to access the data.\n\nTable S2. C-Findings = Indication of impaired organ function due to MC infiltration defining a SM as an aggressive SM.\n\nClick here to access the data.\n\nTable S3. WHO criteria defining Systemic Mastocytosis.\n\nClick here to access the data.\n\n\nReferences\n\nHaenisch B, Nöthen MM, Molderings GJ: Systemic mast cell activation disease: the role of molecular genetic alterations in pathogenesis, heritability and diagnostics. Immunology. 2012; 137(3): 197–205. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfrin LB, Butterfield JH, Raithel M, et al.: Often seen, rarely recognized: mast cell activation disease--a guide to diagnosis and therapeutic options. Ann Med. 2016; 48(3): 190–201. PubMed Abstract \| Publisher Full Text\n\nMolderings GJ: Transgenerational transmission of systemic mast cell activation disease-genetic and epigenetic features. Transl Res. 2016; 174: 86–97. PubMed Abstract \| Publisher Full Text\n\nDroogendijk HJ, Kluin-Nelemans HJ, van Doormaal JJ, et al.: Imatinib mesylate in the treatment of systemic mastocytosis: a phase II trial. Cancer. 2006; 107(2): 345–351. PubMed Abstract \| Publisher Full Text\n\nGotlib J, Kluin-Nelemans HC, George TI, et al.: Efficacy and safety of midostaurin in advanced systemic mastocytosis. N Engl J Med. 2016; 374(26): 2530–2541. PubMed Abstract \| Publisher Full Text\n\nVerstovsek S, Tefferi A, Cortes J, et al.: Phase II study of dasatinib in Philadelphia chromosome-negative acute and chronic myeloid diseases, including systemic mastocytosis. Clin Cancer Res. 2008; 14(12): 3906–3915. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVega-Ruiz A, Cortes JE, Sever M, et al.: Phase II study of imatinib mesylate as therapy for patients with systemic mastocytosis. Leuk Res. 2009; 33(11): 1481–1484. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamaki K, Yoshino S: Tyrosine kinase inhibitor sunitinib relieves systemic and oral antigen-induced anaphylaxes in mice. Allergy. 2012; 67(1): 114–122. PubMed Abstract \| Publisher Full Text\n\nMolderings GJ, Haenisch B, Brettner S, et al.: Pharmacological treatment options for mast cell activation disease. Naunyn Schmiedebergs Arch Pharmacol. 2016; 389(7): 671–694. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAfrin LB, Cichocki FM, Patel K, et al.: Successful treatment of mast cell activation syndrome with sunitinib. Eur J Haematol. 2015; 95(6): 595–597. PubMed Abstract \| Publisher Full Text\n\nCarlisle B, Demko N, Freeman G, et al.: Benefit, Risk, and Outcomes in Drug Development: A Systematic Review of Sunitinib. J Natl Cancer Inst. 2016; 108(1): pii: djv292. PubMed Abstract \| Publisher Full Text\n\nSeidel H, Molderings GJ, Oldenburg J, et al.: Bleeding diathesis in patients with mast cell activation disease. Thromb Haemost. 2011; 106(5): 987–989. PubMed Abstract \| Publisher Full Text\n\nVysniauskaite M, Hertfelder HJ, Oldenburg J, et al.: Determination of plasma heparin level improves identification of systemic mast cell activation disease. PLoS One. 2015; 10(4): e0124912. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "29333", "date": "08 Jan 2018", "name": "Michael Huber", "expertise": [ "Reviewer Expertise Biochemistry and molecular immunology", "mast cell biology", "signal transduction" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nSystemic mastocytosis as one form of systemic mast cell activation disease usually is an incurable disease, which due to patient-specific genetic and/or epigenetic alterations requires a highly personalized therapy to mitigate consequences of mast cell proliferation and/or inflammatory activation. This also implies that a few patients might profit from a therapy, which usually is referred to as noneffective for the majority of patients. Therefore, for the practitioner the publication of such rare cases is important to allow for a potentially effective, though unusual therapy option.\nThe present case report by Molderings et al. offers a detailed description of a patient with aggressive systemic mastocytosis treated with the tyrosine kinase inhibitor (TKI) sunitinib as well as an objective statement about pros and cons of the treatment. Moreover, this case report offers important background knowledge on the current classification of primary mastocytosis (Suppl. Table S1), a list of C-findings = indications of impaired organ function due to mast cell infiltration defining a systemic mastocytosis as an aggressive systemic mastocytosis (Suppl. Table S2), and the WHO 2008 diagnostic criteria for systemic mastocytosis (Sull. Table S3). These enable the reader to access this information important for the comprehension of this case report without obtaining the original literature.\n\nThe reviewer suggests the following changes/additions:\n\na) In the description of the TKI sunitinib, the publication by Todd et al (Todd JR, Becker TM, Kefford RF, Rizos H. Secondary c-Kit mutations confer acquired resistance to RTK inhibitors in c-Kit mutant melanoma cells. Pigment Cell Melanoma Res. 2013; 26: 518-526) should be added. It describes a melanoma cell subline with an additional T670I mutation in c-Kit, which shows resistance to the TKIs imatinib, nilotinib, and dasatinib, but responds to sunitinib.\n\nb) In Suppl. Table S3 (minor Criteria), the sentence “3. Activating KIT mutation at codon 816 in in marrow, blood, or extracutaneous organ(s)“ should be changed to “3. Activating KIT mutation at codon 816 in marrow, blood, or extracutaneous organ(s)“\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "33390", "date": "10 May 2018", "name": "Leonard Weinstock", "expertise": [ "Reviewer Expertise I am actively diagnosing and treating patients with mast cell activation syndrome." ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIs the background of the case’s history and progression described in sufficient detail?\n\nThe background in the Introduction section nicely details the challenges treating systemic mastocytosis. This disease has considerable morbidity based on mediator secretion by malignant mast cells. The discussion clarifies why reduction of mast cell burden by other drugs do not necessarily bring about symptomatic relief.\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? The case report second along with the tables provided excellent details that enable the reader to understand the scope of systemic mastocytosis in this subject, the severity of symptoms, and the challenges in treatment. The patient failed standard therapy for this disease and yet had significant relief with a new medical approach. The problematic side effect of gout might have been prevented by use of allopurinol.\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes. Further studies are warranted.\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes. There is excellent detail to understand the management of systemic mastocytosis.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "29334", "date": "14 May 2018", "name": "Vicki Ratner", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFirst, I would like to clarify that I am a clinician, and not a researcher. I have an expertise in mast cells, in particular, mast cells and interstitial cystitis.\n\nI believe that it is very important to publish case reports, particularly on a disease that is relatively rare, and little is known about. I applaud the authors in their efforts. This case report focuses on a patient with aggressive systemic mastocytosis (ASM). The drug used for treatment was subitinib, which has been approved by the FDA for the treatment of renal cell carcinoma and imitinib–resistant gastrointestinal stromal tumors.\n\nSunitinib, an oral medication, is a multitargeted receptor tyrosine kinase (RTK) inhibitor, as opposed to imitinib, which is also an RTK inhibitor, but can only target one type of receptor. 20% of patients do not respond to imitinib, and 50% become resistant to this drug over time.\n\nThe most common side-effects of sunitinib include HTN, fatigue, stomatitis, neutropenia and thrombocytopenia. Other adverse events include weakness, diarrhea, nausea, anorexia, erythrocytosis (an increase in the mass of the RBC’s) and yellow discoloration of the skin. In addition, the patient may develop hand-foot disease, also called acral erythema, where the palms of the hands and the soles of the feet become red, swollen, numb and cause the skin to peel off. Side-effects in patients using sunitinib occurred in 10% or less of cases, with the exception of HTN, which occurred with much higher frequency.\n\nThere has been only one previous successful case report using sunitinib for aggressive systemic mastocytosis in the European literature. Molderings, et al., reported that the patient experienced less fatigue, which made daily ADL’s (activities of daily living) easier for the patient to perform. In addition, after treatment, the subcutaneous fibrous nodules became movable (as opposed to firmly attached to the underlying tissue) and the smaller nodules disappeared completely. It is unclear why the remaining nodules, now movable, became tender.\n\nIn this second case report, where sunitinib was also used to treat progressive aggressive systemic mastocytosis, there were some benefits. The authors pointed out that the patient had complete resolution of his intense bleeding gums within just 3 days of treatment, and also reported less fatigue. The subcutaneous nodules also became movable and tender. However, the patients’ body hair became white, and there was a decrease in the number of thrombocytes and total protein in the blood. The uric acid level increased to the point of causing gout. At this point, the authors agreed that the side effects outweighed the benefits, and the medication was discontinued.\n\nThere is one case report published by L. Afrin in 2015 about the use of sunitinib in a patient with Mast Cell Activation Syndrome (MCAS) here in the U.S. The patients’ symptoms have completely resolved as long as she remains on the drug, and thus far she has had no side effects. It has been over a year now.\n\nThe cost of sunitinib (Pfizer) is extremely high - almost $150,000 per year. Often, in the U.S., insurance companies have refused to pay for all or part of the costs of this drug, so the cost to the patient can be a tremendous burden. Sunitinib most likely costs less if the dose is lowered and used cyclically. Cyclical use (for example 4 weeks on followed by 2 weeks off) may actually decrease the side effects more than a lower daily dose. However, though cyclical use may be sufficient to control malignancy, it is unlikely that cyclical use would be sufficient to control dysfunctional mast cells, as in MCAS, which might quickly revert to a more symptomatic baseline state of inappropriate activation as soon as the plasma concentration of the drug drops below a therapeutic threshold.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2182
https://f1000research.com/articles/6-1699/v1	18 Sep 17	{ "type": "Research Note", "title": "Utility of massive open online courses (MOOCs) concerning outbreaks of emerging and reemerging diseases", "authors": [ "Guido Bendezu-Quispe", "Junior Smith Torres-Roman", "Brenda Salinas-Ochoa", "Akram Hernández-Vásquez", "Guido Bendezu-Quispe", "Junior Smith Torres-Roman", "Brenda Salinas-Ochoa" ], "abstract": "The emergence and re-emergence of infectious diseases such as Ebola, chikungunya, and Zika increase the necessity of knowledgeable and skilled health professionals. Massive open online courses (MOOCs) arise as opportunities that allow people around the world to participate in higher education courses. A search was conducted on specialized MOOC platforms to find courses related to outbreaks, using terms included in the list of the WHO disease outbreaks from January 1st to December 31st, 2016. We found seven courses about Ebola, two about Zika, three about the dynamics of epidemics and pandemics, and only one course about dengue, chikungunya, and malaria. Most of the courses were conducted in English. The courses on Ebola, Zika and chikungunya were released after their last outbreak. MOOCs could be used to learn about health issues of global relevance, and with the necessity of fast divulgation of knowledge and skills. Translating the courses into more languages could give these courses more traction, and allow participation of professionals in regions affected by these outbreaks.", "keywords": [ "education", "public health professional", "education", "distance", "health education", "education", "medical", "continuing", "disease outbreaks", "hemorrhagic fever", "Ebola", "Zika virus", "chikungunya fever" ], "content": "Introduction\n\nThe emergence and re-emergence of infectious diseases is partly due to the climate change, more specifically due to the rise in global temperature, as well as the increased migration and unplanned urbanization1. These events of great relevance to global health have turned these unknown diseases into realities many health professionals have to face daily.\n\nEbola virus causes an acute and severe disease that is usually fatal if left untreated. Its last outbreak in March 2014 was the largest in history, causing a dramatic number of more than 11,000 deaths and 28,000 new infections worldwide. It affected several countries in West Africa, generating much concern worldwide due to its estimated 50% mortality rate. Similarly, diseases such as chikungunya2 and Zika3 have shown several reasons to be considered serious infectious diseases.\n\nGiven the pandemic potential of these viral diseases, it is important to assess the knowledge and awareness of our health professionals regarding the mode of transmission of these diseases. In this era of globalization and technology, one of the main impacts of the Internet and the web 2.0 have been the acceleration of the process of sharing information, allowing health professionals to have quick and easy access to the latest research in medicine and health4.\n\nMassive open online courses (MOOCs) are online classes or lectures accessible for people all around the world that want to participate in higher education courses. MOOCs material includes videos, slideshows, discussion boards, quizzes, audios or any combination thereof. Usually, participants do not pay any fee to take a course. The topics in MOOCs vary widely and include science, engineering, and arts; and are usually developed by well-known figures in the study area5.\n\n\nMethods\n\nFrom 1st May to 31st May, 2017, we conducted a manual search on several learning platforms that offer MOOCs, including Coursera, edX, FutureLearn, Udacity, Miríada X, Alison, FUN.MOOC, Canvas Network, and Iversity to find courses about disease outbreaks using the terms included in the list of WHO disease outbreaks from January 1st to December 31st, 2016 (Box 1). Information about the learning platform, institution, course length, time required per week, language and subtitles availability for every course were collected and reported using frequencies in the case of categorical data and ranges for numerical data.\n\nepidemic(s), pandemic(s), outbreak(s), emerging diseases, re-emerging diseases, Ebola virus disease, Ebola virus, Ebola, ébola, EVD, viral hemorrhagic fevers, hemorrhagic fever syndrome, Zika, Zika virus, Chikungunya, Chikungunya virus, Lassa, Lassa Virus, MERS-CoV, Middle East respiratory syndrome coronavirus, Yellow fever, Lassa fever, Human infection with avian influenza, Oropouche virus, Rift Valley fever and Wild polio and vaccine derived polio\n\nSource: WHO, Emergencies preparedness, response. Diseases outbreaks by year, 2016. http://www.who.int/csr/don/archive/year/2016/en/\n\n\nResults\n\nWe found seven courses about Ebola, two about Zika, three about the dynamics of epidemics and pandemics, and only one course about dengue, chikungunya, and malaria. The duration of the courses ranged from 2 to 10 weeks. 11 out of 13 courses were held only in English, with the possibility to select subtitles in English or other languages; there was one in English and Chinese, and only one exclusively in Spanish. Most courses (5 out of 13) originated from to USA centers including Emory University, University of Pittsburgh, The Pennsylvania State University, Harvard University and University System of Maryland. The information provided with the courses included audiovisual material, papers, and self-assessments. All the courses presented information about epidemiology and lessons about the outbreaks and prevention activities for a possible new scenario of transmission of infectious diseases. The courses on Ebola, Zika and chikungunya were released after the last outbreaks of these diseases, respectively (Table 1).\n\n\nDiscussion\n\nFinding MOOCs about Ebola, chikungunya, and Zika after the start of their last outbreaks demonstrates the interest of institutions in offering information to the public. The vast majority of courses are offered in English, with a few having subtitles in other languages. MOOCs offer a recent and emerging form of education. There is a continuous increase in the number of courses offered in this format and, by 2015, a total of 35 million participants in 4.200 MOOCs were counted, with 8.27% of these courses corresponding to health and medicine.\n\nThe spread of diseases makes it necessary to invest in alternative methods of spreading knowledge, to improve the capabilities of health professionals in topics that affect people worldwide. MOOCs could be used to learn about health issues of global relevance, and with the necessity of fast divulgation of knowledge and skills. Because the countries most affected by these diseases do not have English as the native language, promoting the translation of content into more languages could give these courses more traction, and allow participation of professionals in regions affected by these outbreaks.\n\n\nData availability\n\nDataset 1: Data on 2016 massive open online courses about disease outbreaks, offered on learning platforms, retrieved from manual searches. The dataset contains information on the learning platform, institution, course length, time required per week, language and availability of subtitles. DOI, 10.5256/f1000research.12639.d1778546.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKilpatrick AM, Randolph SE: Drivers, dynamics, and control of emerging vector-borne zoonotic diseases. Lancet. 2012; 380(9857): 1946–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeaver SC, Lecuit M: Chikungunya virus and the global spread of a mosquito-borne disease. N Engl J Med. 2015; 372(13): 1231–9. PubMed Abstract \| Publisher Full Text\n\nGulland A: Zika virus is a global public health emergency, declares WHO. BMJ. 2016; 352: i657. PubMed Abstract \| Publisher Full Text\n\nLupiáñez-Villanueva F, Mayer MA, Torrent J: Opportunities and challenges of Web 2.0 within the health care systems: an empirical exploration. Inform Health Soc Care. 2009; 34(3): 117–26. PubMed Abstract \| Publisher Full Text\n\nHew KF: Promoting engagement in online courses: What strategies can we learn from three highly rated MOOCS. Br J Educ Technol. 2016; 47(2): 320–41. Publisher Full Text\n\nBendezu-Quispe G, Torres-Roman JS, Salinas-Ochoa B, et al.: Dataset 1 in: Utility of massive open online courses (MOOCs) concerning outbreaks of emerging and reemerging diseases. F1000Research. 2017. Data Source" }	[ { "id": "26029", "date": "29 Sep 2017", "name": "Mohamed A Gouda", "expertise": [ "Reviewer Expertise Medical education", "oncology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a nice short report from the study conducted by Bendezu-Quispe G et al. about the existence of MOOCs with focus on emerging and re-emerging diseases. The idea is simple, and so is the methodology. However, I have concern regarding the time frame selected for the study which was limited to the year 2016. Some of the outbreaks highlighted in the background started in earlier years and were terminated in 2016. I think that more valid and strong data could have been provided if there were no time limits thus allowing for exploration of MOOCs presented at the time of epidemics. Otherwise, authors have properly presented their data. I believe their work would be a good start for further exploration of this area.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3289", "date": "19 Dec 2017", "name": "Akram Hernández-Vásquez", "role": "Author Response", "response": "Dear Dr. GoudaThank you for your comments regarding our Research note on MOOCs on emerging and reemerging diseases. About the time frame, in February 2016, WHO declared Zika’s outbreak a public health emergency of international concern. Based on that announcement, we conducted a search about MOOCs of Zika. We expected that both public and academia concern about this disease will impact the number of MOOCs developed about this topic. However, the presence of outbreaks of diseases such as chikungunya and Oropuche during 2016, expanded our research scope. The 2016 WHO’s list of diseases outbreaks included: Ebola, viral hemorrhagic fevers, hemorrhagic fever syndrome, Zika, Chikungunya, Lassa, MERS-CoV, Middle East respiratory syndrome coronavirus, Yellow fever, Human infection with avian influenza, Oropouche virus, Rift Valley fever and Wild polio and vaccine derived polio. This list includes a vast number of diseases that were also included in the annual lists of outbreaks published by WHO before 2016. Hence, it is not expected that a significant increase in the number of MOOCs after increasing the list of outbreaks before 2016." } ] }, { "id": "28591", "date": "05 Dec 2017", "name": "Shirley Ann Williams", "expertise": [ "Reviewer Expertise Learning technologies", "MOOCs" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting research note identifying MOOCs relating to emerging and re-emerging diseases. The work in its current form is acceptable as a research note, and the conclusion that such MOOCs need to be available in more languages is an important point.\nIf the authors are to extend their work there is scope for improvement:\nThe search window was very limited, it is possible that MOOCs that had completed would not be found. Adding MOOC aggregators (such as Class Central) to the search areas could have identified more courses, than those on offer in May 2017.\n\nThere are many barriers to accessing MOOCs, especially in developing countries, for example access to computers, connectivity, digital skills, as well as fluency in international languages. Consideration of all potential barriers will be important in future work.\n\nThe target audience of the MOOCs is not clear in this report, and there may be benefit in classifying courses that are aimed at health professionals and those aimed at the general public. Then considering whether there is benefit in a direct linguistic translation for particular MOOCs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3288", "date": "27 Dec 2017", "name": "Akram Hernández-Vásquez", "role": "Author Response", "response": "Dear Dr. Williams and Dr. LiyanagunawardenaThank you very much for your response. Regarding your comments:1. About the use of MOOC aggregators, the authors used Class Central and MOOC List for both obtain the list of MOOC websites and for obtaining information about MOOCs completed before our time frame. Now, that information is included in methods:From 1st May to 31st May 2017, we conducted a manual search on several learning platforms that offer MOOCs, including Coursera, edX, FutureLearn, Udacity, Miríada X, Alison, FUN.MOOC, Canvas Network, and University to find courses about disease outbreaks using the terms included in the list of WHO disease outbreaks from January 1st to December 31st, 2016 (Box 1). If the information about the course was not available on the learning platform that originally offered it, we use the information provided by MOOC aggregator platforms such as Class Central and MOOC List because Information about the learning platform, institution, course length, time required per week, language and subtitles availability for every course were collected and reported using frequencies in the case of categorical data and ranges for numerical data.2. Thank you for your recommendation. Our study group is developing a survey to measure barriers to accessing and using MOOCs produced locally.3. About the target population, all the courses were aimed at health professionals. Considering this, we have included this information in the results section:We found seven courses about Ebola, two about Zika, three about the dynamics of epidemics and pandemics, and only one course about dengue, chikungunya, and malaria. The duration of the courses ranged from 2 to 10 weeks. 11 out of 13 courses were held only in English, with the possibility to select subtitles in English or other languages; there was one in English and Chinese and only one exclusively in Spanish. Most courses (5 out of 13) originated from to USA centers including Emory University, University of Pittsburgh, The Pennsylvania State University, Harvard University and University System of Maryland. The information provided with the courses included audiovisual material, papers, and self-assessments. All the courses were made for health-related professionals and presented information about epidemiology and lessons about the outbreaks and prevention activities for a possible new scenario of transmission of infectious diseases. The courses on Ebola, Zika, and chikungunya were released after the last outbreaks of these diseases, respectively." } ] } ]	1	https://f1000research.com/articles/6-1699
https://f1000research.com/articles/6-1560/v1	24 Aug 17	{ "type": "Software Tool Article", "title": "TicTimer software for measuring tic suppression", "authors": [ "Jonathan K. Black", "Jonathan M. Koller", "Kevin J. Black", "Jonathan K. Black", "Jonathan M. Koller" ], "abstract": "Woods and Himle developed a standardized tic suppression paradigm (TSP) for the experimental setting, to quantify the effects of intentional tic suppression in Tourette syndrome. The present article describes a Java program that automates record keeping and reward dispensing during the several experimental conditions of the TSP. The software can optionally be connected to a commercial reward token dispenser to further automate reward delivery to the participant. The timing of all tics, 10-second tic-free intervals, and dispensed rewards is recorded in plain text files for later analysis. Expected applications include research on Tourette syndrome and related disorders.", "keywords": [ "tic disorders", "Tourette syndrome", "reward", "reinforcement", "psychology", "software" ], "content": "Introduction\n\nWoods and Himle developed a tic suppression paradigm (TSP) that could be used in the experimental setting to demonstrate and quantify the effects of intentional tic suppression on tic rate in Tourette syndrome (TS) and other tic disorders1–4. In this paradigm, each participant is observed during several experimental conditions, baseline and differential reinforcement of zero-rate ticcing (DRO), and sometimes also verbal instruction to suppress tics and noncontingent reinforcement (NCR).\n\nIn the course of conducting a longitudinal study of children with Provisional Tic Disorder5, we found that tic suppression is seen within the first few months after a child’s first tic6. We also found that the TSP required substantial investigator effort, and we started writing software with the following goals:\n\nautomated tic counting, timing and record-keeping;\n\nautomated reward delivery in the DRO condition;\n\nautomated reward delivery in the NCR condition.\n\nThe overall motivations included not only convenience but also improvement in accuracy. We present the software here to facilitate its use by others.\n\n\nMethods\n\nTicTimer first has the user set up the details for a session, then it runs a clock for the specified session time while writing significant events to a log file. The program writes a line to the log file for each of the following events: session started and ended, tic detected, ten seconds passed without tics, and reward dispensed. Each line includes the time of the event. By parsing through each line in a log file, a python script can extract and summarize the key data.\n\nThe hardware allows reward tokens to be dispensed automatically to the study participant. One end of the long cable enters the token dispenser and its two wires attach to the two pins in the Passive Connection Panel that, when shorted, trigger release of a reward token. The Student Trainer Interface box provides power to the token dispenser box and a remote pushbutton for manually triggering token release. The other end of the long cable connects to the two normally open pins on the relay inside of the small plastic box. The USB to TTL serial cable attaches to the input pins on the relay, with the USB end of the cable leaving the box to attach to the computer running TicTimer. Figure 1 shows the final assembly.\n\nParts list:\n\nMed Associates token dispenser box, Part #ENV-703\n\nMed Associates SG-595 Student Trainer Interface\n\nMed Associates SG-215D3 Passive Connection Panel\n\n5V Relay module (SainSmart part # 20-018-100)\n\nUSB to TTL serial cable (Adafruit part # 954)\n\nPlastic project enclosure box (Velleman part # WCAH2855)\n\n~6m cable with at least one pair of wires free\n\nSystem software requirements are Java 8 and RXTX for Java, a library for serial port communication. Binaries for Windows and Linux are provided by fizzed.com. Python 3 was used for the log file reader script.\n\nThe program can be run with or without the relay and USB cable. If the hardware is set up and connected to the computer, the program can start in “link mode.” Otherwise, TicTimer can still be run in “button mode,” in which the automatic reward system is replaced by a human who presses the push button attached to the Student Trainer Interface when prompted by a beep and a red flash on the computer screen.\n\nThe following procedure applies to both reward modes. First, the user presses “Setup” to choose which type of session is being run and to specify where the session log should be saved. For the NCR condition, the user is also prompted to identify the log file from a previously completed DRO session in the same subject (which provides the timing for the rewards dispensed in the NCR condition). Once setup is completed, the session can be started. During a session, the person observing the subject records tics by clicking the “Tic Detected” button or by pressing “T” or the space bar. If the session type includes rewards (DRO and NCR), they are dispensed appropriately. The session ends when the predetermined time elapses or when the user ends it manually by pressing “End Session” or by closing the window.\n\nIf a session is ended manually and restarted, the new session log will be appended to the old one unless a new file was chosen in setup. If a log file contains multiple sessions, only the last session will be used by the NCR mode (which requires a DRO session file in setup) and the data reader script.\n\nTo summarize the data from a TicTimer log file, run the accompanying python script (TT_Data.py) with one or more log files as arguments. For each log file given, the script reports the length of the session and the number of tics, 10-second tic-free intervals, and rewards dispensed during the session.\n\n\nUse cases\n\nWe have provided 8 sample session log files as Supplementary Files (Supplementary files 1–Supplementary file 8) (subject100_session_TicTimer_log.txt). These are examples of the files that TicTimer creates during a session. Each line in a log file contains an event and the time at which it occurred. Log files are written in plain English, so they can be read directly if desired. These sample data originated from a participant in the study described by Greene et al.6, but all identifying data were removed and these files no longer comprise human subjects data.\n\nThe file TT_Data_output.txt (Supplementary file 9) contains output from the python script, summarizing those session files. This output is again in plain text, reporting the session length and the number of each type of event recorded for each log file.\n\n\nConclusions\n\nThe TicTimer program, now connected to the reward token dispenser, has simplified implementing the TSP and improved the accuracy of reward delivery (given inevitable limitations of human attention and response time in button mode). The software, while designed for our purposes in tic disorder research, may find other uses. The most obvious of these may be for research on traditional habit disorders; for instance, hair pulling and skin picking appear in the “Obsessive-compulsive and related disorders” section of DSM-57.\n\nA preprint of this work has been published on Authorea.com8.\n\n\nSoftware availability\n\nThe source code is available on GitHub under a BSD 3-clause license.\n\nThe current release is available on Zenodo, at DOI 10.5281/zenodo.837884.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSoftware development and manuscript preparation were funded in part by the U.S. National Institutes of Health (NIH), grant numbers K24 MH087913, R21 NS091635, and R01 MH104030.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe gratefully acknowledge the initial tic suppression studies by Woods and Himle.\n\n\nSupplementary material\n\nThe first 8 supplementary files are the output from running 8 TicTimer sessions in one subject.\n\nSupplementary File 1: Baseline Log 1\n\nThis file came from the first baseline session.\n\nClick here to access the data.\n\nSupplementary File 2: Verbal Log 1\n\nOutput from the first session with only verbal instruction to resist tics.\n\nClick here to access the data.\n\nSupplementary File 3: DRO Log 1\n\nOutput from the first DRO session.\n\nClick here to access the data.\n\nSupplementary File 4: NCR Log 1\n\nOutput from the first NCR session.\n\nClick here to access the data.\n\nSupplementary File 5: Verbal Log 2\n\nOutput from the second session with only verbal instruction to resist tics.\n\nClick here to access the data.\n\nSupplementary File 6: Baseline Log 2\n\nOutput from the second baseline session.\n\nClick here to access the data.\n\nSupplementary File 7: DRO Log 2\n\nOutput from the second DRO session.\n\nClick here to access the data.\n\nSupplementary File 8: NCR Log 2\n\nOutput from the second NCR session.\n\nClick here to access the data.\n\nSupplementary File 9: Data Reader Script Output\n\nOutput from the command \"python TT_Data.py _log.txt\" in the directory containing the 8 sample log files. It summarizes the data contained in those files.\n\nClick here to access the data.\n\n\nReferences\n\nWoods DW, Himle MB: Creating tic suppression: comparing the effects of verbal instruction to differential reinforcement. J Appl Behav Anal. 2004; 37(3): 417–20. PubMed Abstract \| Free Full Text\n\nHimle MB, Woods DW: An experimental evaluation of tic suppression and the tic rebound effect. Behav Res Ther. 2005; 43(11): 1443–51. PubMed Abstract \| Publisher Full Text\n\nHimle MB, Woods DW, Bunaciu L: Evaluating the role of contingency in differentially reinforced tic suppression. J Appl Behav Anal. 2008; 41(2): 285–9. PubMed Abstract \| Free Full Text\n\nLyon GJ, Samar SM, Conelea C, et al.: Testing tic suppression: comparing the effects of dexmethylphenidate to no medication in children and adolescents with attention-deficit/hyperactivity disorder and Tourette’s disorder. J Child Adolesc Psychopharmacol. 2010; 20(4): 283–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBlack KJ, Black ER, Greene DJ, et al.: Provisional Tic Disorder: What to tell parents when their child first starts ticcing [version 1; referees: 3 approved]. F1000Res. 2016; 5: 696. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGreene DJ, Koller JM, Robichaux-Viehoever A, et al.: Reward enhances tic suppression in children within months of tic disorder onset. Dev Cogn Neurosci. 2015; 11: 65–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAmerican Psychiatric Association: Obsessive-Compulsive and Related Disorders. In: Diagnostic and Statistical Manual of Mental Disorders. Fifth Edition. Arlington, VA: American Psychiatric Association; 2013. Publisher Full Text\n\nBlack JK, Koller JM, Black KJ: TicTimer software for measuring tic suppression. Authorea, 2017. Publisher Full Text" }	[ { "id": "27424", "date": "30 Oct 2017", "name": "Danielle C. Cath", "expertise": [ "Reviewer Expertise Science practioner", "specialised in research and clinics of diagnosis and treatment of tics and Tourettes Disorder" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis tool might be very useful in clinical practice to make tic suppression more objectively measured, and to substantiate the direct results of tic suppression practicing. It is potentially a useful addition to current practice of CBT in tic suppression, and entails a worthwhile initiative to develop this tool.\nHowever, I miss information on some aspects:\nDirect comparison with other potential ways to generate objective quantifiable results; for instance the advantages compared to video-based tic counting.\n\nI miss reflection on potential disadvantages of the tool, and limitations of the tool (for instance in practicing in daily life, not only in the lab, is unclear.\n\nIn the plain text output, I miss summary information across sessions, and f.i. graphs on tic course across sessions that could be presented to the patient and discussed. The latter would be really helpful for patients and therapist\n\nThe device can in no way be used in more naturalistic sessions outside the treatment room, the authors should speculate about this, and about future developments to make the tool more practical and applicable in different situations and contexts.\n\nI miss pilot information on direct comparisons between conventional tic counting and the counting with the aid of the tictimer.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3278", "date": "22 Dec 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We thank Prof. Cath for her thoughtful comments. We failed to clarify adequately the primary purpose of this software tool. It was designed rather narrowly for use in a standardized Tic Suppression Protocol in a clinical research office setting. This fact affects many of the specific comments below. We have revised the Introduction to address this point.Here are responses to each item in your review: comparing to other tic counting methods: This tool can be used (and we have used it) to facilitate counting tics on video recordings. The main drawback with using the software to count and time tics on video recordings is that one cannot give “live” feedback in the Differential Reinforcement of Other (zero-rate ticcing) condition. limitations: We have rewritten the introduction to clarify the limited intended purpose, and clarify in Discussion that use outside of the research office will require adaptations. graphs showing changes over time, for clinical use: This is a useful feature that may be added in the future. In the meantime, we point out that the information in Supplementary File 9 can be imported easily into a spreadsheet program for creating such graphs. can't be used in more naturalistic settings: Correct. See our initial comment, above. We have in fact adapted some features of TicTimer for a web-based tool intended for more naturalistic (home-based) tic suppression timing.[1] comparison of TicTimer to conventional tic counting: We do not feel this is necessary, because (as we apparently failed to explain adequately) in each case an expert human is watching the person with tics. TicTimer adds the (more or less) precise timing of each tic to the record. [1] Black JK and Black KJ. Software for web-based tic suppression training [version 1; referees: awaiting peer review]. F1000Research 2017, 6:2150 (doi: 10.12688/f1000research.13460.1)" } ] }, { "id": "26009", "date": "06 Nov 2017", "name": "Patrick Haggard", "expertise": [ "Reviewer Expertise Neurocognition of human sensorimotor function" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a simple but functional piece of software for logging tics, and automating a reward schedule based on them.\n\nThe hardware/software combination will be useful for researchers and possibly for behavioural analysis/clinical training – though this publication does not aim to describe clinical outcomes.\n\nThe software does not appear to actually detect tics automatically – there is no interface to accelometry for example. Rather an examiner seems to have to observe the examinee constantly, and enter manually when a tic occurs, presumably by pressing a button. However, this requirement is unfortunately not specified directly in the text. It also means there is a subjective component: how does the examiner judge what is a tic and what is, for example, a voluntary action: these are tricky questions which face researchers in this area.\n\nThe data is logged in a simple way that is clearly explained, and suitable for postprocessing using standard packages – but the user seems to have to write their own scripts to produce synthetic reports.\n\nThe package logs occurrence of a 10 s period in which no tic is reported – but this time window appears arbitrary: it would be useful (a) to justify it, and (b) to allow the user to change it, either at run time, or in subsequent sensitivity analysis of the log files: how long a period of tic-free behaviour should count as a success event?\n\nThe strength of the tool seems to be the automation of reward scheduling for studies of voluntary tic suppression in children.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [ { "c_id": "3279", "date": "22 Dec 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We are grateful for the careful attention and thoughtful suggestions of Prof. Haggard. Here we reply to each point in his review of version 1. The software doesn't detect tics automatically; the accuracy of any rater-identified tics depend on the skill of the rater. These points are all valid. The scope of the software is limited, primarily being useful to track and time the tics as noted by an expert observer, who indeed logs each tic by pressing a keyboard button or clicking a mouse. We have updated the text to clarify these details. \"The user seems to have to write their own scripts to produce synthetic reports.\" We do supply (via GitHub) the python script TT_Data.py for summarizing each primary data file, but apparently we failed to provide a direct link to this script. We have corrected this omission in this revision. The choice of 10 seconds as the tic-free interval of interest is arbitrary. We chose the 10s period because of its prior use in the studies cited, but you’re correct that this choice is otherwise arbitrary. We have explained this choice in the revised ms. The suggestions in (b) are interesting, but to some extent outside of our plan for the software. We have modified the source code, however, to make a change in tic-free intervals easier to make at compile time. \"The strength of the tool seems to be the automation of reward scheduling for studies of voluntary tic suppression in children.\" Yes, we also see that as one of its key advantages. We expect TicTimer could be applied equally well to studies of adults." } ] } ]	1	https://f1000research.com/articles/6-1560
https://f1000research.com/articles/6-1997/v1	13 Nov 17	{ "type": "Research Note", "title": "The impact of fresh gas flow on wash-in, wash-out time and gas consumption for sevoflurane and desflurane, comparing two anaesthesia machines, a test-lung study.", "authors": [ "Fredrik Leijonhufvud", "Fredrik Jöneby", "Jan G. Jakobsson", "Fredrik Leijonhufvud", "Fredrik Jöneby" ], "abstract": "Low-flow anaesthesia is considered beneficial for the patient and the environment, and it is cost reducing due to reduced anaesthetic gas consumption. An initial high-flow to saturate the circle system (wash-in) is desirable from a clinical point of view. We measured the wash-in and wash-out times (time to saturate and to eliminate the anaesthetic agent, AA), for sevoflurane and desflurane, in a test-lung with fixed 3 MAC vaporizer setting at different fresh gas flow (FGF) and calculated the consumption of AA. We tried to find an optimal flow rate for speed and gas consumption, comparing two anaesthesia machines (AMs): Aisys and Flow-i. Time to reach 1 minimal alveolar concentration (MAC) (wash-in) decreased (p<0.05) at higher flow rates (1 – 2 – 4) but plateaued at 4-4.8 l/min. The consumption of AA was at its lowest around 4-4.8 l/min (optimal flow) for all but the Aisys /desflurane group. Wash-out times decreased as FGF increased, until reaching plateau at FGF of 4-6 l/min. Aisys had generally shorter wash-in times at flow rates < 4 l/min as well as lower consumption of AA. At higher flow rates there were little difference between the AMs. The “optimal FGF” for wash-out, elimination of gas from the test-lung and circle system, plateaued with no increase in speed beyond 6 l/min. A fresh gas flow of 4 l/min. seems “optimal” taking speed to reach a 1 MAC ET and gas consumption into account during wash-in with a fixed 3 MAC vaporizer setting, and increasing fresh gas flow beyond 6 l/min does not seem to confirm major benefit during wash-out", "keywords": [ "wash-in", "wash-out", "low-flow anaesthesia", "fresh gas flow", "inhalational anaesthetics", "sevoflurane", "desflurane" ], "content": "Introduction\n\nLow flow anaesthesia is associated with several benefits, reducing the heat loss caused by cold gases and improving humidification in the airways1. It is also environmentally friendly, reducing the release of anaesthetic agents into the atmosphere, and lastly, it also reduces costs. The initial period needed to achieve a steady state is dependent on fresh gas flow and the vaporizer setting2. Many different FGF schemes have been described for the wash-in3,4. The wash-out of the inhaled anaesthetic from the lungs and circle is also of importance, to facilitate a rapid awakening and recommencement of protective reflexes.\n\nThe aim of the present study was to measure the time required to reach stable end tidal 1 MAC anaesthetic (wash-in), to measure gas consumption during wash-in and the time needed to eliminate the anaesthetic agent (wash-out) for sevoflurane and desflurane, and to compare the impact of different fresh gas flows and anaesthesia machines.\n\n\nMethods\n\nThis study used a test-lung setup. It was conducted in the operating room with proper scavenging equipment and central gas supply at Danderyds hospital, Sweden.\n\nTwo standard anaesthesia machines were used: Flow-iR (Maquet Critical Care AB, Solna, Sweden) and AisysR (GE Healthcare, Madison, WI, USA), and two anaesthetic agents: sevoflurane and desflurane, making up a total of four test groups. All standard safety measures were followed, including checking each machine for performance and leakage before every session, in accordance with the Instruction for use (IFU), to ensure a safe working environment.\n\nA standard disposable adult circle system was used (GE patient circuit, adult, disposable, 1.8 m and 1.5 litre volume), including a Y-piece at the end of the tube. A humidity-filter (Humid-vent, filter compact A) was fitted on the Y-piece. The circle system also included a standard CO2 absorber (soda lime canister) even though no carbon dioxide was used in the experiments, because some of the anaesthetic agent (AA) may also be absorbed and therefore mimic clinical conditions better.\n\nA test lung was assembled using the following equipment: The Maquet test-lung 190 (Maquet Critical Care AB, Solna, Sweden), tidal volume max 1 litre and internal volume of 0, was mounted on one of the tubes of another Y-piece. Two 2-liter Intersurgical reservoir bags were connected to a T-tube to the other tube of the Y-piece.\n\nMeasurements of FiAA and EtAA of sevoflurane and desflurane were done with a mainstream sensor (instead of the AM side-stream sensors). IRMA AX +, (Masimo Sweden AB, Danderyd, Sweden) with an accuracy of 0.15 vol. % was used. This allowed us to get more exact measurements without any delay or dilution of gases. The mainstream sensor was connected to an external computer via a USB port and the Gasmaster® (Masimo Sweden AB, Danderyd, Sweden) program was used to record our measurements.\n\nDuring the entire experiment a fixed respiratory rate and tidal volume was used, set at 12/min and 400 ml respectively. The positive end-expiratory pressure (PEEP) was set at 5 cm H2O and the inspiratory/expiratory rate was set two 1:2.\n\nTwo direct injection vaporizers were used for the Flow-i (one for each AA) and two variable bypass vaporizers for the Aisys (one for each AA). The AAs were released at fixed vaporizer settings of 3 × MAC-value (6 % and 18 % for sevoflurane and desflurane respectively).\n\nThe timer/data log was started at the same time as when the vaporizer was turned on. The wash-in time to reach 1 MAC (i.e. increasing the EtAA concentration from 0 to 1 MAC), adjusted for a 40-year-old, 70 kg male (2 % for sevoflurane and 6 % for desflurane), was measured three consecutive times for each fresh gas flow (FGF). The FGF tested included 1, 2, 4, 4.75, 4.8, 6 and 8 l/min, with a composition of 80 % oxygen and 20 % air for each of the AAs and anaesthesia machines.\n\nWash-out (reducing EtAA to 0 MAC) was performed after each wash-in session of the test-lung/circle. This was done by turning off the vaporizer and setting the FGF to one of 5 (2, 4, 6, 8 and 10 l/min) on each anaesthesia machine (AM) and AA.\n\nThe same protocol was used for sevoflurane and desflurane as well as for Flow-i and Aisys and the results for each wash-in session were presented as a mean of 3.\n\nGas consumption was calculated using the known wash-in time, FGF, vaporizer settings (VA concentration) and the vapour/liquid quote for each AA: vapour (ml)/liquid (ml) = 184 and vapour (ml)/liquid (ml) = 210 for sevoflurane and desflurane respectively. It means that 1ml of liquid AA equals 184ml and 210ml of vaporized sevoflurane and desflurane, respectively5.\n\nThis gives the equation: AA consumption = time(s)×FGFx100060×VA conc (vol%)(vapor/liquidquote)×100 (vol%)\n\nSPSS 24 (IBM, Armonk, NY, USA) was used for statistical analysis. All wash-in data was presented as mean of 3 measurements with standard deviation (SD) calculated. The mean time to reach 1 MAC for the different FGF was compared among the Flow-i and Aisys AM using analysis of variance (ANOVA). To determine which of the mean FGFs differed significantly from each other, we also performed Tukey's post hoc test. A p< 0.05 was considered statistically significant.\n\n\nEthical statement\n\nThis is a test model study. The research does not involve human participants and/or animals, and thus no informed consent has been requested. The set-up is entirely experimental and no human or animals have been exposed to anaesthetics, and thus no ethical review board assessment has been considered necessary, according to Swedish research regulations.\n\n\nResults\n\nWash-in times were significantly faster with higher flow rates for FGF spanning from 1, 2 and 4 l/min (p < 0.05) for all 4 groups (Figure 1 and Figure 2). There was however a plateau starting at FGF 4–4.75/4.8 l/min, where increasing the FGF further up to 6 or 8 l/min did not shorten the time to reach 1 MAC significantly; similar patterns could be observed in all 4 groups. With Flow-i that plateau started at 4 and 4.8 l/min for sevoflurane and desflurane, respectively. With Aisys this plateau started at 4 l/min, for both the sevoflurane and desflurane group. Aisys had generally shorter wash-in times for both sevoflurane and desflurane at flow rates < 4 l/min. For flow rates of 4 l/min and above the difference was very small but somewhat more pronounced for desflurane wash-in, with Flow-i having shorter wash-in times than Aisys (Figure 1 and Figure 2).\n\nAnaesthetic agent consumption showed a somewhat different pattern (Figure 3). The lowest consumption differed between sevoflurane and desflurane and also between the Aisys and Flow-i. The lowest consumption of AA per wash-in for Flow-i sevoflurane and Flow-i/desflurane, 1.4ml and 3.9ml respectively, was calculated at FGF equal to minute ventilation (4.8 l/min). The lowest consumption of AA for Aisys /sevoflurane, 1.3 ml, was calculated at a FGF at 4 l/min. The lowest consumption of AA for Aisys /desflurane, 3 ml, was calculated at the lowest FGF (1 l/min), in difference to the other 3 groups, followed by FGF 2 and 4 l/min with a calculated consumption of AA of 3.3 ml and 3.9 ml respectively. This was unique for Aisys /desflurane (Figure 3 A–D).\n\n(A) Flow-i /sevoflurane. (B) Flow-i /desflurane. (C) Aisys /sevoflurane. (D) Aisys/desflurane. Time(s) is on the Y-axis. FGF (l/min) is on the X-axis. AA (ml) is on the secondary Y-axis.\n\n(A) sevoflurane (B) desflurane. Time (s) is on the Y-axis and FGF (l/min) is on the X-axis.\n\nWash-out times were faster at higher FGF until a plateau was reached at around FGF 4–6 l/min. Increasing the FGF further did not lead to faster wash-out times, except for the Aisys / sevoflurane group. The fastest wash-out time with Flow-i /sevoflurane, 349s, was recorded at FGF 6 l/min where it also plateaued. Wash-out times for Flow-i /desflurane plateaued at FGF 4 l/min and 1013s, which differed a few seconds from the fastest wash-out time, 1007s, recorded at FGF 8 l/min. Wash-out times for Aisys /sevoflurane had a plateau that was not as distinct as the other groups with the fastest wash-out time, 303s, recorded at FGF 10 l/min. The fastest wash-out time for Aisys /desflurane, 797s, was recorded at FGF 6 l/min, where it reached a clear plateau (Table 1). There was minor difference in elimination, Aisys being marginally faster (Figure 4 A–B). No statistical analysis was performed on the wash-out data.\n\n\nDiscussion\n\nWe found, in this test-lung setup, that it is possible to increase the anaesthetic agent concentration from 0 to 1 MAC value in around one minute by using a FGF of 4–4.8 l/min on both AMs, but raising the FGF further did not result in shorter wash-in times. These flow rates were also the most efficient, in terms of both speed and gas consumption for both anaesthetic agents (sevoflurane and desflurane) and anaesthetic machines (Aisys and Flow-i) except when using Aisys and desflurane. Aisys had generally shorter wash-in times when using lower flow rates (< 4 l/min). For flow rates of 4 l/min and above, the difference was very small between the two machines tested. Anaesthetic agent consumption showed different patterns, but Aisys had generally lower gas consumption than Flow-i for both AAs. The shortest wash-out times were found at FGFs of 4–6 l/min; raising the FGF further did not result in shorter wash-out times. Wash-out of desflurane from the test-lung was more than twice as time-consuming as the sevoflurane wash-out.\n\nThis is merely a test-lung study and it was expected that there would be more rapid wash-in with increased fresh gas flow, but it is important to acknowledge the plateau at around 4–5 l/min FGF. Our results also show that Maplesons mathematical calculations of a theoretical optimal FGF at around 4 l/min were accurate6. The result is also in line with what Shin et al. found in their study: increasing the FGF from low flow to moderately high flow will decrease the wash-in time7. However, our results show that there seem to be no benefits to raising the FGF to levels of 6 l/min and above while performing a wash-in.\n\nLikewise the finding of increased AA consumption at both low and high FGFs is of interest, minimising the consumption of anaesthetic vapour is of economical as well as ecological importance. There was a difference between the AMs in terms of which FGF was most effective. We noted that the most effective FGF for the Flow-i, in terms of both consumption of AA and shortest wash-in time, was equal to the minute volume – 4.8 l/min. This applied to both sevoflurane and desflurane wash-in.\n\nThe small difference in wash-in between the AMs at lower fresh gas flows are in line with our previous study with a simpler test setup with sevoflurane8. We used a more “physiologic” test-lung setup in the present study, which we believe mimics functional residual capacity and tidal volume for a 70 kg male. Our test-lung was constructed to represent both tidal volume (the volume of a normal breath) and the FRC in our test-lung setting, making the measurements more realistic. The Maquet test-lung 190 was representing the tidal volume and the two reservoir bags were representing a normal FRC (4 litre) in a 40-year-old male9. We used an external main stream multi gas sensor in the present study, possibly reducing the difference in monitor performance.\n\nThe finding that gas consumption is higher with desflurane than with sevoflurane may not be surprising, when taking the difference in MAC gas concentration into account. The huge difference, the twice as long time to eliminating desflurane as compared to sevoflurane, is to us however unexpected.\n\nThere are several limitations to this study, indeed this is merely a test-lung experiment and one may of course argue about its clinical application. The vaporizer setting was fixed at 3 MAC. We believe that our findings have clinical implications; we know that high fresh gas flows do not provide major benefits during wash-in or wash-out and there is the possibility to wash-in to a 1 MAC concentration within about a minute, 4 – 5 l/min FGF being the “optimal” time for wash-in. Both Aisys and Flow-i have built-in techniques for target end-tidal MAC, were fresh gas and vaporizers are automatically set to reach the target value10.\n\n\nConclusions\n\nWash-in times with fixed 3 MAC vaporizer settings decreased as the FGF increased, but plateaued at around flow rates of 4–4.8 l/min (optimal flow) on both AM. It is more effective, in terms of consumption of AA, to use optimal flow than lower or higher flow rates. Wash-out times plateaued at around flow rates of 4–6 l/min, higher FGF than that does not produce faster wash-out times. Further studies are required to confirm our findings in clinical practice, but also to study the end tidal target algorithms available on the AMs.\n\n\nData availability\n\nDataset 1: Raw data for the measurements: Flow-I sevoflurane, Flow-I desflurane, Aisys sevoflurane and Aisys desflurane. DOI, 10.5256/f1000research.13064.d18380611", "appendix": "Competing interests\n\n\n\nJan Jakobsson has been paid for lecturing and taking part in advisory boards for Maquet.\n\n\nGrant information\n\nThis study has been supported by the Department of Anaesthesia at Danderyds Hospital.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nMaquet provided assistance with an anaesthetic machine for the experimental setup. Mazimo provided a main stream multi-gas monitor.\n\n\nReferences\n\nBrattwall M, Warrén-Stomberg M, Hesselvik F, et al.: Brief review: theory and practice of minimal fresh gas flow anesthesia. Can J Anaesth. 2012; 59(8): 785–97. PubMed Abstract \| Publisher Full Text\n\nHorwitz M, Jakobsson JG: Desflurane and sevoflurane use during low- and minimal-flow anesthesia at fixed vaporizer settings. Minerva Anestesiol. 2016; 82(2): 180–5. PubMed Abstract\n\nHendrickx JF, Dewulf BB, De Mey N, et al.: Development and performance of a two-step desflurane-O2/N2O fresh gas flow sequence. J Clin Anesth. 2008; 20(7): 501–7. PubMed Abstract \| Publisher Full Text\n\nSathitkarnmanee T, Tribuddharat S, Nonlhaopol D, et al.: 1-1-12 one-step wash-in scheme for desflurane low flow anesthesia: performance without nitrous oxide. Drug Des Devel Ther. 2015; 9: 977–81. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBiro P: Calculation of volatile anaesthetics consumption from agent concentration and fresh gas flow. Acta Anaesthesiol Scand. 2014; 58(8): 968–72. PubMed Abstract \| Publisher Full Text\n\nMapleson WW: The theoretical ideal fresh-gas flow sequence at the start of low-flow anaesthesia. Anaesthesia. 1998; 53(3): 264–72. PubMed Abstract \| Publisher Full Text\n\nShin HW, Yu HN, Bae GE, et al.: The effect of fresh gas flow rate and type of anesthesia machine on time to reach target sevoflurane concentration. BMC Anesthesiol. 2017; 17(1): 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJakobsson P, Lindgren M, Jakobsson JG: Wash-in and wash-out of sevoflurane in a test-lung model: A comparison between Aisys and FLOW-i [version 2; referees: 2 approved, 1 approved with reservations]. F1000Res. 2017; 6: 389. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoch B, Friedrich N, Völzke H, et al.: Static lung volumes and airway resistance reference values in healthy adults. Respirology. 2013; 18(1): 170–8. PubMed Abstract \| Publisher Full Text\n\nDe Medts R, Carette R, De Wolf AM, et al.: Desflurane usage during anesthesia with and without N2O using FLOW-i Automatic Gas Control with three different wash-in speeds. J Clin Monit Comput. 2017; 1–7. PubMed Abstract \| Publisher Full Text\n\nJakobsson JG, Fredrik L, Fredrik J: Dataset 1 in: The impact of fresh gas flow on wash-in, wash-out time and gas consumption for sevoflurane and desflurane, comparing two anaesthesia machines, a test-lung study. F1000Research. 2017. Data Source" }	[ { "id": "27920", "date": "20 Nov 2017", "name": "Katarina Hallen", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me for a peer review.\nI have some questions to the authors regarding the study: The impact of fresh gas flow on wash-in, wash-out time and gas consumption for sevoflurane and desflurane, comparing two anaesthesia machines, a test-lung study. by J. Jakobsson.\n\nIs there a difference between the two vaporizer used in the experiment? Comment on the difference in a direct-injection and a bypass affect how respirators works. Why the maximum inflow of the two gases? This is not clinically relevant. Would not it be better for another group with half the inflow, 4% and 8%? Why non-parametric tests? Is this normal distribution of data? The figures are not sufficient quality, high solution pictures please. Explain in an educational way how you calculate gas consumption. Do you have any theory why Aisys is faster at low flow with desflurane? Do you have any theories? Why does it take longer to wash-out desflurane than sevoflurane in a mechanical lung model? This does not correspond to the clinical picture. Does desflurane have lower density than sevoflurane? Is that why it takes longer to wash-out the desfluran? Can you come up with some theories about the most remarkable result in the study? I can not find sevoflurane wash-out in Figure 4.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? I cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3209", "date": "27 Nov 2017", "name": "Jan Jakobsson", "role": "Author Response F1000Research Advisory Board Member", "response": "Thank you for valid comments. Please find below responses to your queries. Is there a difference between the two vaporizer used in the experiment? Yes indeed both the technique to create the gaseous phase, the internal reservoir and the respiratory phase when gas is introduced into fresh gas. The Flow-i “injects gas” during inspiration while the Aysis has a more traditional vaporizer technique. The internal gas volume, reservoir is smaller in the Flow-i. This is the reason for our interest in comparing the machine, to assess how these different solution create difference during a rapid wash-in, gas bolus. Comment on the difference in a direct-injection and a bypass affect how respirators works. The aim of the study was indeed to study how these different techniques act in a test-lung set up at different flow rates. It would also be interesting to study lower vaporizer settings at that study may be worth carrying out. Why the maximum inflow of the two gases? This is not clinically relevant. Would not it be better for another group with half the inflow, 4% and 8%? The aim of this study was to study the bolus, high concentration performance. It would indeed be of interest to study lower setting. Why non-parametric tests? Is this normal distribution of data? Traditional paremetric test was used, ANOVA test. The figures are not sufficient quality, high solution pictures please. We will improve image Explain in an educational way how you calculate gas consumption. The formula is derived from the reference. Do you have any theory why Aisys is faster at low flow with desflurane? Do you have any theories? This may be an effect of the different vaporizer techniques and the phase during inspiration/expiration gas is delivered to the fresh gas. Why does it take longer to wash-out desflurane than sevoflurane in a mechanical lung model? This does not correspond to the clinical picture. The finding is also to us somewhat surprising. One must acknowledge that this is merely the lung volume and circle wash-out. The low blodd gas solubility impacts the biological wash-out from the human body. It does not describe the elimination from volumes per see. Does desflurane have lower density than sevoflurane? Is that why it takes longer to wash-out the desfluran? Can you come up with some theories about the most remarkable result in the study? The longer wash-out may be related merely to the huger amount of gas, the 6 % gas volume. I can not find sevoflurane wash-out in Figure 4. The Figure 4 A. presents sevoflurane wash-out." } ] }, { "id": "27918", "date": "12 Dec 2017", "name": "Sirirat Tribuddharat", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis report is interesting and may have clinical relevant in routine practice. However, the authors should address to the following points:\nA figure showing the diagram of the circuit assembly including the test lung should be included. It’s quite confusing about the number of ‘Y-piece’. Normally, the test lung should be attached to the humidifier-filtered connected to a Y-piece. Why using 2 reservoir bags when normally only 1 is used in routine practice? Where is the location of the main stream sensor?\n\nPlease quote a reference for ‘1 MAC adjusted of a 40 year-old (2% for sevoflurane and 6% for desflurane). It’s quite different from “Nickalls RWD. BJA 2003; 91: 170-4”.\n\nWe have both anesthetic machines. The flowmeters on both Flow-i and AISYS are digitally controlled. They can be adjusted with accuracy of only 1 decimal place of litre, i.e., 0.1 L/min. Although the display on AISYS shows 2 decimal placed, i.e., 2.00 L/min, we can adjust flow of 0.1 L/min at a time only. How can the authors set FGF at 4.75 L/min?\n\nThe caption of Figures 3 and 4 does not match the figure, e.g., (a) in figure but (A) in caption.\n\nThe wash-in time derived from a test lung may not be inferred to human lung. The major confounding factor of wash-in time is anesthetic agent uptake from lung into human body ‘three compartments’ which affected by blood gas partition coefficient, cardiac output, organ tissue flow, organ tissue volume, and blood-tissue partition coefficient. That makes sevoflurane having longer wash-in time at same MAC and FGF. The test lung model did not take into account these factors. The authors should address to this point.\n\nThe wash-out time has the same problem. The test lung did not consider amount of anesthetic agent uptake into the body which has to be eliminated out of the body into account. Normally, at the same MAC, sevoflurane has longer wash-out time compared with desflurane because of higher blood-gas, blood-tissue and blood-fat solubility. The authors should address to this point.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3281", "date": "22 Dec 2017", "name": "Jan Jakobsson", "role": "Author Response F1000Research Advisory Board Member", "response": "This report is interesting and may have clinical relevant in routine practice. However, the authors should address to the following points: A figure showing the diagram of the circuit assembly including the test lung should be included. It’s quite confusing about the number of ‘Y-piece’. Normally, the test lung should be attached to the humidifier-filtered connected to a Y-piece. Why using 2 reservoir bags when normally only 1 is used in routine practice? Where is the location of the main stream sensor? A graph of the set-up is added Please quote a reference for ‘1 MAC adjusted of a 40 year-old (2% for sevoflurane and 6% for desflurane). It’s quite different from “Nickalls RWD. BJA 2003; 91: 170-4”. We used the FDA approved USP desflurane information sheet and Baxter Summary of Product characteristics; the 1 MAC for 40-45 years adult is set at 6 % (www.baxter.com.pr/healthcare_professionals/.../suprane.html). Likewise for sevoflurane we used the FDA and Baxter product documentations, stating 1 MAC at aye of 40 being 2.1 % but we used for this study the MAC 2 and 6 %. We have both anesthetic machines. The flowmeters on both Flow-i and AISYS are digitally controlled. They can be adjusted with accuracy of only 1 decimal place of litre, i.e., 0.1 L/min. Although the display on AISYS shows 2 decimal placed, i.e., 2.00 L/min, we can adjust flow of 0.1 L/min at a time only. How can the authors set FGF at 4.75 L/min? The flow setting should be with 1 decimal, the 4.75 is incorrect for the Flow-I corrected The caption of Figures 3 and 4 does not match the figure, e.g., (a) in figure but (A) in caption. Corrected excuse the edit mistake The wash-in time derived from a test lung may not be inferred to human lung. The major confounding factor of wash-in time is anesthetic agent uptake from lung into human body ‘three compartments’ which affected by blood gas partition coefficient, cardiac output, organ tissue flow, organ tissue volume, and blood-tissue partition coefficient. That makes sevoflurane having longer wash-in time at same MAC and FGF. The test lung model did not take into account these factors. The authors should address to this point. This is indeed merely an experimental test-lung set-up and we cannot comment on the human uptake and elimination, e.g. the impact of the different blood gas solubility for the agents tested. The wash-out time has the same problem. The test lung did not consider amount of anesthetic agent uptake into the body which has to be eliminated out of the body into account. Normally, at the same MAC, sevoflurane has longer wash-out time compared with desflurane because of higher blood-gas, blood-tissue and blood-fat solubility. The authors should address to this point. These, comments 6 & 7, are indeed one of limitations with our study, it is merely a test lung study. We have addressed the limitation in the discussion section, and we also clearly state that further studies are warranted confirming our results in the clinical setting. We still believe that our study provide information around the machines, how the workstations performs during wash-in and wash-out of the gaseous phases. The uptake, blood solubility and likewise elimination must of course be taken into account." } ] }, { "id": "28449", "date": "15 Dec 2017", "name": "Moritz A. Kretzschmar", "expertise": [ "Reviewer Expertise Kinetics of volatile anesthetics", "ventilation/perfusion relationship of the lung", "thoracic anesthesia" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this interesting article. It is wonderful to see research on basic science addressing questions which are often taken for granted in clinical practice.\n\nWe do have a few questions:\n\nWhat is the clinical value of comparing sevoflurane and desflurane at the same MAC, if just the machines are of interest (and the solubility and clinical effects are negligible), would it not have been better to use the same vol%?\n\nThere are only two factors influencing wash-in to the circuit, and those are circuit volume and fresh gas flow. Setting aside the patient at the end of the anesthesia machine, this study evaluates the wash-in/-out of the total apparatus dead space. To better compare the measurements from the Aisys and the Flow-I, it would be necessary to reference the volumes (bag/volume reflector, internal tubing, respective soda lime canister etc.) of both machines up to the y-piece. We presume they were not identical, but by how much do they differ?\n\nThe wash-in time plateaued at around minute ventilation, which was to be expected, as both systems could be considered almost closed, with only minimal leakage. For the wash-out, the lowest FGF that eliminates rebreathing should indicate the plateau. Whereas with sevoflurane there is no apparent difference between the machines, reflecting the relationship between circle volume and FGF, the difference with desflurane is quite interesting. Could some materials used in the Flow-i have absorbed desflurane – We are thinking of the volume reflector or valves?\n\nWe cannot completely follow the data files: if the main stream sensor was placed after the y-piece, the high data points reflect Fi of the agent. Why was this position chosen and not the proximal (to the y-piece) end of the expiratory tube. This could have eliminated inspiratory/expiratory mixing in the acquisition, as the values were not recorded synchronized to the breath cycle.\n\nAn illustration of the experimental setup could enhance understanding for the reader.\n\nThe information of Figures 1 and 2 are redundant – they are repeated in Fig 3.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3280", "date": "22 Dec 2017", "name": "Jan Jakobsson", "role": "Author Response F1000Research Advisory Board Member", "response": "We do have a few questions: What is the clinical value of comparing sevoflurane and desflurane at the same MAC, if just the machines are of interest (and the solubility and clinical effects are negligible), would it not have been better to use the same vol%? The aim of the study was to compare the anaesthetic works-station and the two third generation in haled anaesthetics, aiming for an similar end-tidal 1 MAC gas concentration and subsequently time to wash-out has from these gas volumes. There are only two factors influencing wash-in to the circuit, and those are circuit volume and fresh gas flow. Setting aside the patient at the end of the anesthesia machine, this study evaluates the wash-in/-out of the total apparatus dead space. To better compare the measurements from the Aisys and the Flow-I, it would be necessary to reference the volumes (bag/volume reflector, internal tubing, respective soda lime canister etc.) of both machines up to the y-piece. We presume they were not identical, but by how much do they differ? The present study was set up to gain insight to whether the machine different in time need to saturate internal and externa has reservoirs, including an anticipated normal adult lung volume. The wash-in time plateaued at around minute ventilation, which was to be expected, as both systems could be considered almost closed, with only minimal leakage. For the wash-out, the lowest FGF that eliminates rebreathing should indicate the plateau. Whereas with sevoflurane there is no apparent difference between the machines, reflecting the relationship between circle volume and FGF, the difference with desflurane is quite interesting. Could some materials used in the Flow-i have absorbed desflurane – We are thinking of the volume reflector or valves? We are not able to assess potential losses. Both machines are “tested” and approved for use with both sevoflurane and desflurane. We cannot completely follow the data files: if the main stream sensor was placed after the y-piece, the high data points reflect Fi of the agent. Why was this position chosen and not the proximal (to the y-piece) end of the expiratory tube. This could have eliminated inspiratory/expiratory mixing in the acquisition, as the values were not recorded synchronized to the breath cycle. The main-stream gas monitor was attached in parallel with in side-stream nipple. The readings, the recorded and presented values were the highest and stable values at each time-point. An illustration of the experimental setup could enhance understanding for the reader. A graph of the set-up is added The information of Figures 1 and 2 are redundant – they are repeated in Fig 3. Deleted as suggested" } ] } ]	1	https://f1000research.com/articles/6-1997
https://f1000research.com/articles/6-2169/v1	22 Dec 17	{ "type": "Research Note", "title": "Early language development in preterm children without neurological damage: a longitudinal study", "authors": [ "Micaela Capobianco", "Luca Cerniglia", "Micaela Capobianco" ], "abstract": "Children born at a very low gestational age, even those without neurosensory damages, are at risk of linguistic disorders. This longitudinal study aimed at analyzing communicative and language abilities in preterm children during their second year of life, through a standardized questionnaire, with particular attention to the communicative and language abilities that predict the first verbal skills. Our results showed that preterm children are slower than full-terms in language acquisition particularly at earlier stages of development. The differences between the two groups of children was significant only at 16 and 18 months. Preterms use more simplistic linguistic categories for longer than full-terms, with regards to lexicon composition and syntactic complexity. This different pattern could involve more qualitative, rather than quantitative, aspects of developmental processes that characterize language acquisition in preterms and full-term children.", "keywords": [ "Preterm children", "biological risk", "linguistic disorders", "first verbal skills" ], "content": "Introduction\n\nThe study of early language development processes in children who were born at biological risk, such as preterm infants without neurosensory damages, is crucial. In fact, it has been shown that children born at very low gestational age are at risk of linguistic disorders in their first years of life (Capobianco et al., 2010; Cimino et al., 2016). The present longitudinal study aimed at analyzing communication and language abilities in preterm children during their second year of life, through a standardized questionnaire for parents, the “Primo Vocabolario del Bambino” (Caselli et al., 2015) (PVB, Italian version of “MacArthur-Bates”, age 8–36 months) with particular attention to communication abilities that are predictors of the first verbal skills at 3 years (Pizzuto & Capobianco, 2008; Capobianco et al., 2017).\n\n\nMethod\n\n40 children participated in this study: 20 preterm children (7 females; 13 males) and 20 full-term children (7 females; 13 males). Inclusion criteria for preterm children were: a) No neurological damages at birth; b) APGAR between 7 and 10; c) Gestational age between 31 and 33 weeks; d) Weight appropriate for the gestational age. Preterm children had an average gestational age of 32 weeks (s.d.=2) and an average weight at birth 2200 gr. (s.d.=250). Inclusion criteria for full-term children were: a) No neurological damages at birth; b) Gestational age between 37 and 40 weeks; c) weight appropriate for the gestational age; d) no previous history of neurodevelopmental disorders. The average gestational age of full-terms was 39 weeks (s.d=3) and their weight at birth was 3500 gr. (s.d=300). All preterm and full-term children had a normal IQ (>85) evaluated by the Bayley Scales (II version) at 18 and 24 months of age. All participants were from an upper-middle-class family (calculated with an ad hoc questionnaire assessing the educational level and occupational status of parents). The preterm group and the full-term group were matched on age and sex.\n\nPreterm children were recruited at the University Hospital of Rome, Policlinico Umberto I, (Puericulture Clinic), where all children at risk (including preterm children born with no neurological damage) undergo a protocol starting at birth for the monitoring of cognitive and language development every 3 months. Full-term children were selected from a larger longitudinal study on the spontaneous productions collection during the third year of age (Capobianco e Devescovi, 2008; Capobianco et al., 2017).\n\nAll parents of children recruited for this study gave their written consent for participation in this research and for the publication of its results. The study was approved by the Ethical Committee of Sapienza, University of Rome (ID: 1/2007).\n\nThe language ability of preterm and full-term children was examined at 16, 18, 20, 22 and 24 months of age through the Italian version of the MacArthur-Bates Communicative Development Inventories (MB-CDI questionnaire) (Caselli et al., 2015): at 16 months “Words and Gesture“ was used, Complete Form (validated for Italian toddlers of age from 8 to 24 months); at 18, 20, 22 and 24 months of age the other version labeled “Words and Phrases” was used (validated for Italian toddlers age from 18 to 36 months). Families of preterm children were asked to fill in the questionnaire during the clinical follow-up in Hospital. Families of full-term children were asked to fill in the questionnaire at their home every time the researcher went to the child’s home to collect the spontaneous production data.\n\nAt 16 months the following indexes were derived by PVB questionnaire: word comprehension, word production (with lexicon composition: proportions of nouns, verbs and functors) gesture production. At 18, 20, 22 and 24 months the following measures were derived: word productions, lexicon composition (proportions of nouns, verbs and functors), number of phrases used and syntactic complexity with respect to the proportion of phrases classified as “with functional words” (e.g. “mommy car”) vs. “without functional words” (e.g. “the car of mommy”) used.\n\nParametrical analysis T-test (t Student) and ANOVA (Analysis of Variance) were conducted to analyze the differences in language abilities between ages within each group (preterm ad full-term infants).\n\n\nResults\n\nAt 16 months the difference between pre-terms and full-terms was significant for word comprehension [t(38) =2.19, p<0.05] and gesture production [t(38)=3.79, p<0.01]. Examining lexicon composition (proportions) in comprehension at 16 months, we found that full-term children showed a higher percentage of Verbs (t (22)= -2.24, p= 0.03) and Functors (t (22)= -1.16, p= 0.07) than pre-terms. The differences between pre-terms and full-terms was significant for Verbs [t(38) = 2.16, p<0.05] and Nouns [t(38)=-2.08, p<0.05], but not for Functors [t(38)=0.931, n.s.] in comprehension.\n\nResults showed that preterms produced a lower number of words then full-term children at all assessment points (16, 18, 20, 22 and 24 months), although in the normative range of typical development (PVB). Moreover, the statistical analysis (ANOVA) showed a significant growth of lexicon production over time in both preterms and full-term children [F4,35=15.6, p<0.01]. The differences between preterms and full-terms was significant at 16 months [t(38)=4,05, p<0.01] and 18 months [t(38)=2.43, p<0.05], but not at subsequent assessment points (20, 22, 24 months). We also found that preterm children use less Verbs and Functors at all age. Nouns increased over time, and full-term children show a systematic increase of Verbs and Functors from 18 to 24 months, and a decrease of Nouns over time [20 months: t(38)=2.25, p<0.05; 22 months: t(38)=2.29, p<0.05].\n\nFigure 1 shows the number of phrases produced by preterm children and full-terms at 18, 20, 22 and 24 months. Figure 2a and the Figure 2b show respectively the number of phrases with and without functional words produced by preterm children and full-terms at 18, 20, 22 and 24 months. Preterm children used a lower number of phrases than full-terms at all ages. Statistical analysis (ANOVA), however, showed a significant growth of phrase production over time in both preterms and full-terms [F3.36=12.12, p<0.01]. The differences were significant only at 18 months [t(38)=2.80, p<0.05] but not at subsequent assessment points [F3.36=12.12, p<0.01]. At 24 months (last observation) we found that preterm and full-term children produced a similar number of phrases [preterm=13.9 phrases; full-term=13.2 phrases]. We found significant differences in the use of phrases classified as “with and without functional words” (Figure 2a and Figure 2b) [F(3.36)=6.29, p<0.01]. However, the data showed that preterms used more phrases classified as “without functional words” at all age and phrases classified as “with functional words” were produced only at 22 months (14.7 %), increasing at 24 months (25.9 %). In contrast, full-terms used the phrases classified as “with functional words” at the first observation (18 months) and the frequency of this category of phrases increased between 20 and 24 months. The use of phrases classified as “without functional words” decreased in full-terms over time. At 24 months full-terms produced significantly more phrases classified as “with functional words” (89.2 %) than phrases classified as “without functional words” (10.8 %).\n\n(a) Phrases “incomplete” and “complete” in preterms from 18 to 24 months (b) phrases “incomplete” and “complete” in full-terms from 18 to 24 months.\n\n\nDiscussions\n\nOur data showed that preterms were slower than full-term children in language acquisition especially at earlier stages of development. Even if preterms had a reduced vocabulary in general, the differences between the two groups of children were significant only at 16 and 18 months. Moreover, preterm children tended to a naturally recover primary acquisitions during the second year of life. The differences were present in the qualitative aspects of language abilities, such as the lexicon and verbal combinations at 24 months. We observed that preterms used more simplistic linguistic categories longer than full-terms, more simplistic linguistic categories referring to lexicon composition (nouns) and syntactic complexity (phrases without functional words). These different patterns could involve more qualitative rather than quantitative aspects of developmental processes that characterize language acquisition in preterms and full-term children. This data have several clinical and research implications. First, it can be useful for the early prevention of language disorders in preterm children, through the screening of the specific elements of lexicon composition and of phrase complexity. Second, they confirmed the importance of longitudinal studies in this field and the usefulness of chronological age in assessing the early language development of preterm children comparing them with full-term offspring, to observe the early recovery and the qualitative differences in language production.\n\n\nData availability\n\nAll raw data is accessible at: http://dx.doi.org/10.17632/zgvk4hb8hb.2 (Cerniglia, 2017)\n\nData are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe authors acknowledge and are grateful to all children and parents participating in the study.\n\n\nReferences\n\nCapobianco M, Pizzuto EA, Devescovi A: Gesture–speech combinations and early verbal abilities. Interact Stud. 2017; 18(1): 55–76. Publisher Full Text\n\nCapobianco M, Riccio G, Devescovi A: Early communicative and language development in preterm infants without neurological damage. J Appl Res Intellect Disabil. 2010; 23(5): 513.\n\nCaselli MC, Casadio P: Il Primo Vocabulario Del Bambino (Children’s early vocabulary). Milan, Italy: Franco Angeli. 1995.\n\nCerniglia L: “Preterm/Fullterm”, Mendeley Data, v2. 2017. Data Source\n\nCimino S, Cerniglia L, Almenara CA, et al.: Developmental trajectories of body mass index and emotional-behavioral functioning of underweight children: A longitudinal study. Sci Rep. 2016; 6: 20211. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPizzuto EA, Capobianco M: Is pointing “just” pointing? Unraveling the complexity of indexes in spoken and signed discourse. Gesture. 2008; 8(1): 82–103. Publisher Full Text" }	[ { "id": "29655", "date": "15 Jan 2018", "name": "Laura Serra", "expertise": [ "Reviewer Expertise Neuropsychology", "psychology", "psychiatry", "neurology", "neuroimaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI have reviewed the Research Note “Early language development in preterm children without neurological damage: a longitudinal study”. I think that the paper is well written and offers a relevant contribution to studies on the early communicative and language development in preterm children without neurological damage. I accept the work with minor revisions. In particular:\nI think it's necessary to add in the Reference list a more recent article on the preterms early language developmental.\n\nThere is a mismatch for the citation of Questionnaire “PVB” (1995 or 2017?) between Reference list and text.\n\nI suggest adding a very brief passage about the reasons why the authors chose those measures over others.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "29991", "date": "29 Jan 2018", "name": "Allison B. Ellawadi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you very much for the opportunity to review this interesting and well written report. My comments for the statistical analysis and discussion are listed below.\nStatistical Analysis:\nWord comprehension and gesture productions – a Multivariate Analysis of Variance using group as the independent variable and gesture and word comprehension as the dependent variables. A follow-up analysis using the different lexical categories would then be appropriate.\n\nIn the Word Production Analyses – the authors note that “We also found that preterm children use less Verbs and Functors at all age. Nouns increased over time, and full-term children show a systematic increase of Verbs and Functors from 18 to 24 months, and a decrease of Nouns over time [20 months: t(38)=2.25, p<0.05; 22 months: t(38)=2.29, p<0.05].” This is a bit confusing. Does this indicate that the preterm infants lost some of the nouns that they were producing (e.g., parents reported that they produced 25 nouns at 20 months but then only 10 nouns at 24 months?). The information in this section appears contradictory with regard to the noun data reported.\n\nDiscussion:\nIt seems to point to the children having initially slower language acquisition but then they must demonstrate a faster rate of growth at some point (or the full term infants demonstrate a slowed rate of growth) since there is not a difference in words and phrases by 24 and the only different in vocab was at 16 and 18 months. While the authors state this later on in the first paragraph, this is not immediately clear. I would recommend starting with something that states that although there are initial delays the preterm infants appear to catch up.\n\nThe authors should put in a couple of more sentences to discuss what is meant by “qualitative aspects” of language abilities and why it mattes that preterms seem to have differences in these aspects of language later in in development.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3386", "date": "30 Jan 2018", "name": "Micaela Capobianco", "role": "Reader Comment", "response": "Thank you very much for your comments!Micaela Capobianco" } ] } ]	1	https://f1000research.com/articles/6-2169
https://f1000research.com/articles/6-1842/v1	16 Oct 17	{ "type": "Research Article", "title": "The use of dexmedetomidine and intravenous acetaminophen for the prevention of postoperative delirium in cardiac surgery patients over 60 years of age: a pilot study", "authors": [ "Ammu T. Susheela", "Senthil Packiasabapathy", "Doris-Vanessa Gasangwa", "Melissa Patxot", "Jason O’Neal", "Edward Marcantonio", "Balachundhar Subramaniam", "Senthil Packiasabapathy", "Doris-Vanessa Gasangwa", "Melissa Patxot", "Jason O’Neal", "Edward Marcantonio", "Balachundhar Subramaniam" ], "abstract": "Background: Delirium is associated with many negative health outcomes. Postoperative sedation and opioid administration may contribute to delirium. We hypothesize that the use of dexmedetomidine and Intravenous acetaminophen (IVA) may lead to reduced opioid consumption and decreased incidence of postoperative delirium. This pilot study aims to assess feasibility of using dexmedetomidine and IVA in cardiac surgical patients, and obtain effect size estimates for incidence and duration of delirium. Methods: A total of 12 adult patients >60 years of age undergoing cardiac surgery were recruited for the study after IRB approval and randomized into 4 groups: Propofol only (P), Propofol with IVA (P+A), Dexmedetomidine only (D), Dexmedetomidine with IVA (D+A). Preoperative baseline cognition and postoperative delirium was assessed daily until discharge. The feasibility was assessed by the number of patients who successfully completed the study. Results: All patients completed the study protocol successfully. The total incidence of delirium in the study population was 42% (5/12): 67% (2/3) in the group P, and 67% (2/3) in the group D, 33% (1/3) in D+A group and 0%(0/3) P+A group. The incidence of delirium was 17% (1/6) in the group receiving IVA compared to 67% (4/6) that did not receive IVA. The mean duration of delirium was 0-1 days. One patient expired after surgery, unrelated to the study protocol. One patient in the D group experienced hypotension with systolic blood pressure <90 mm of Hg. Conclusions: The feasibility of performing a large-scale project is ascertained by the study. Patients receiving IVA had lower incidence of delirium compared to patients not receiving IVA which suggests that IVA may have a role in reducing the incidence of delirium. A prospective randomized, placebo-controlled trial will be the next step in investigating the role of dexmedetomidine and IVA in reducing the incidence of delirium.", "keywords": [ "delirium", "dexmedetomidine", "acetaminophen", "cardiac surgery", "coronary artery bypass grafting (CABG)", "propofol", "cognitive assessments" ], "content": "Introduction\n\nDelirium is defined as a change in mental status, characterized by acute onset and fluctuating course, inattention, disorganized thinking, and altered level of consciousness1,2. Delirium increases the risk of mortality, readmissions, and accelerated cognitive decline2–6. Around 158 billion dollars of national healthcare cost is attributable to delirium7. The incidence of delirium in cardiac surgery is 11–46%2. Delirium is preventable in 30–40% of the cases2. Some of the modifiable risk factors for delirium include the choice of analgesic and sedatives8.\n\nCurrently used sedatives, like midazolam, act via GABA receptors and release deliriogenic mediators9. Dexmedetomidine is an alpha 2 adrenergic agonist, with no interaction with GABA receptors.\n\nA recent study by Li et al. in 285 elderly cardiac surgical patients evaluated dexmedetomidine versus propofol and showed no difference in the incidence of delirium10. A major limitation of this study is that Confusion Assessment Method (CAM) and CAM- ICU are limited assessments and may miss delirium. There are other tools that could help assess cognitive assessment more efficiently. A meta-analysis suggested that the use of dexmedetomidine for sedation in cardiac surgery patients may reduce the incidence of delirium9. The first study in the meta-analysis was a retrospective study by Corbett SM et al. that does not mention how delirium was assessed in individual hospitals11. The second study by Shehabi Y et al. was not powered adequately and used CAM-ICU for assessing delirium both in intubated and extubated patients which have reduced sensitivity for delirium in verbal patients and could have underestimated the incidence of delirium12. The third study was a small study by Maldonado JR et al. of only 118 patients recruited and only 90 patients finally analyzed, with 28 randomized patients excluded for protocol violations which introduce a significant selection bias13. It also showed a 94% reduction in the incidence of delirium and this effect size is almost implausibly large and has not been seen in other studies. The final study by Dasta JF et al. was not designed to identify patients with delirium with any specific tools such as CAM and thus could have gross underestimated delirium incidence14.\n\nThe use of IV acetaminophen (IVA) has been shown in multiple studies to reduce the amount of opioids consumed by patients undergoing surgeries4,15. IVA has never been studied in the context of cardiac surgery and delirium prevention. So, we hypothesized that the use of dexmedetomidine and acetaminophen would provide adequate analgesia, leading to a decreased opioid consumption and decreased incidence of delirium.\n\n\nMethods\n\nThis is a pilot trial which ran from 13th November 2013 to 9th April 2015 to assess the feasibility of using IV dexmedetomidine and acetaminophen in the cardiac surgical intensive care unit. The study also aimed to obtain effect size estimates for primary study outcomes which will help power an ongoing large scale randomized trial (NCT02546765, registered on 13th January 2015).\n\nThis is a single-centered, double-blinded, prospective, randomized controlled pilot trial.\n\nPatients who are 60 years of age or older who were undergoing coronary artery bypass grafting (CABG), and/or valve surgery were included in the study. Exclusion criteria were preoperative left ventricular ejection fraction < 30%, emergent and percutaneous procedures, aortic surgeries, preexisting cognitive impairment, recent seizures, patients on medications for cognitive decline, serum creatinine > 2 mg%, liver dysfunction, known history of alcohol or drug abuse, and hypersensitivity to any of the study drugs. (Figure 1)\n\nFourteen patients were recruited (the initial 2 patients to help train the investigators in the use of cognitive assessments). The remaining twelve patients were randomized into the following 4 groups, containing 3 patients each (1:1:1:1 allocation). (Table 1)\n\nAll groups will receive bolus doses of IV opioids (morphine and hydromorphone) as needed for breakthrough pain. I.V. Acetaminophen will be given to group 1 and group 3 every 6 hours for 48 hours, postoperatively.\n\nEach patient was allotted a randomization number based on which the pharmacist assigned the study medications.\n\nPeri-operative anesthetic management was administered according to standard of care. Intra-operative propofol infusion was administered at the clinician’s discretion for patients in propofol group. In the post-operative period, propofol infusion was titrated to 25–100 µg/kg/min. If the patient was randomized to dexmedetomidine group, propofol infusion was avoided inside the operating room (OR). Dexmedetomidine infusion was given after chest closure at a dose of 0.1–1.0 µg/kg/hr. The medications for sedation were continued in the post-operative period until extubation or for a minimal duration of 6 hours. For patients randomized to IVA group, IVA 1 gram was given every 6 hours for the first 48 hours postoperatively up to a total of eight doses.\n\nCognitive assessments were conducted using standard battery used for Minimental (MMSE) and Confusion Assessment Method (CAM).\n\nThe primary outcome measured was the proportion of patients who completed the protocol successfully, which reflects the feasibility of the study. The incidence of delirium was measured to obtain effect size estimates for future studies. Secondary outcomes included hypotension, duration of delirium, breakthrough analgesic requirements, ICU days, and length of hospital stay.\n\n\nResults\n\nThe baseline characteristics of the patients were comparable in all groups. The total incidence of delirium was 42% (5/12). The incidence of delirium was 67% (2/3) with propofol and 67% (2/3) with dexmedetomidine. Dexmedetomidine+Acetaminophen group had an incidence of 33% (1/3). The Propofol+Acetaminophen group had no occurrence of delirium. Interestingly, only 17% (1/6) of the subjects who received IVA were diagnosed with delirium compared to 67% (4/6) in the group who did not receive IVA. (Table 2) Also, the incidence of delirium was 33% (2/6) in the propofol groups as compared to 50% (3/6) in the dexmedetomidine groups. The mean duration of delirium ranged from 0 to 1 day. (Table 3) Secondary outcomes were similar between the groups. One patient expired after surgery, unrelated to the study protocol. One patient in the dexmedetomidine group experienced a significant hypotension with systolic blood pressure <90 mm of Hg.\n\nOpioid consumption is expressed in hydromorphone equivalent\n\nMean dexmedetomidine dose is expressed as infusion rate of mcg/kg/hr\n\nMean propofol dose is expressed as infusion rate of mcg/kg/min\n\nOpioid consumption is expressed in hydromorphone equivalent\n\n\nDiscussion\n\nThe use of IV dexmedetomidine and acetaminophen in the cardiac surgical patients was feasible. The study protocol was easily incorporated into patient care. All the enrolled patients completed the study protocol. There were no instances where the implementation of the study protocol was abandoned by the physicians. Sedation provided was useful. Apart from the incidence of hypotension in one patient with dexmedetomidine use, no other adverse events recorded directly related to the intervention.\n\nNo other statistical evaluation was done due to the small sample size. The overall incidence of delirium in our trial, at 42%, is close to previous reports2. The incidence of delirium in patients receiving IVA was 17% compared to 67% in the other group suggesting that IVA may be beneficial in reducing the incidence of delirium following cardiac surgery. The pilot study led to a larger ongoing trial (NCT02546765).\n\nA major limitation of our study is the small sample size, but this was a feasibility trial to study effect size for the design of future larger studies. Also, there was no blinding for the choice of sedatives used in the patients. The assessors were blinded to acetaminophen administration.\n\nA multi-center randomized, controlled trial will be the next step in investigating the role of dexmedetomidine and IVA in reducing the incidence of delirium.\n\n\nData availability\n\nF1000Research: Dataset 1. The datasets used and/or analyzed during the current study. No statistical evaluation was done due to the small sample size., 10.5256/f1000research.12552.d18082816\n\n\nEthics and consent\n\nThis study has been approved by the Committee on Clinical Investigations at Beth Israel Deaconess Medical Center (IRB Protocol #: 2013-P-000149). Informed consent was obtained for all subjects prior to initiation of study procedures. The study was conducted from 13th November 2013 to 9th April 2015.", "appendix": "Competing interests\n\n\n\nThe authors declare that they have no competing interests of conflicts of interest.\n\n\nGrant information\n\nBalahundhar Subramaniam is supported by the National Institutes of Health Research Project Grant GM 098406.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nBellelli G, Morandi A, Di Santo SG, et al.: “Delirium Day”: a nationwide point prevalence study of delirium in older hospitalized patients using an easy standardized diagnostic tool. BMC Med. 2016; 14(1): 106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInouye SK, Westendorp RG, Saczynski JS: Delirium in elderly people. Lancet. 2014; 383(9920): 911–922. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInouye SK: Delirium in older persons. N Engl J Med. 2006; 354(11): 1157–1165. PubMed Abstract \| Publisher Full Text\n\nO'Neal JB: The utility of intravenous acetaminophen in the perioperative period. Front Public Health. 2013; 1: 25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSaczynski JS, Marcantonio ER, Quach L, et al.: Cognitive trajectories after postoperative delirium. N Engl J Med. 2012; 367(1): 30–39. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCui V, Tedeschi CM, Kronzer VL, et al.: Protocol for an observational study of delirium in the post-anaesthesia care unit (PACU) as a potential predictor of subsequent postoperative delirium. BMJ Open. 2017; 7(7): e016402. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeslie DL, Marcantonio ER, Zhang Y, et al.: One-year health care costs associated with delirium in the elderly population. Arch Intern Med. 2008; 168(1): 27–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRudolph JL, Jones RN, Levkoff SE, et al.: Derivation and validation of a preoperative prediction rule for delirium after cardiac surgery. Circulation. 2009; 119(2): 229–36. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLin YY, He B, Chen J, et al.: Can dexmedetomidine be a safe and efficacious sedative agent in post-cardiac surgery patients? a meta-analysis. Crit Care. 2012; 16(5): R169. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi X, Yang J, Nie XL, et al.: Impact of dexmedetomidine on the incidence of delirium in elderly patients after cardiac surgery: A randomized controlled trial. PLoS One. 2017; 12(2): e0170757. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCorbett SM, Rebuck JA, Greene CM, et al.: Dexmedetomidine does not improve patient satisfaction when compared with propofol during mechanical ventilation. Crit Care Med. 2005; 33(5): 940–5. PubMed Abstract \| Publisher Full Text\n\nShehabi Y, Grant P, Wolfenden H, et al.: Prevalence of delirium with dexmedetomidine compared with morphine based therapy after cardiac surgery: a randomized controlled trial (DEXmedetomidine COmpared to Morphine-DEXCOM Study). Anesthesiology. 2009; 111(5): 1075–84. PubMed Abstract \| Publisher Full Text\n\nMaldonado JR, Wysong A, van der Starre PJ, et al.: Dexmedetomidine and the reduction of postoperative delirium after cardiac surgery. Psychosomatics. 2009; 50(3): 206–17. PubMed Abstract \| Publisher Full Text\n\nDasta JF, Jacobi J, Sesti AM, et al.: Addition of dexmedetomidine to standard sedation regimens after cardiac surgery: an outcomes analysis. Pharmacotherapy. 2006; 26(6): 798–805. PubMed Abstract \| Publisher Full Text\n\nMacario A, Royal MA: A literature review of randomized clinical trials of intravenous acetaminophen (paracetamol) for acute postoperative pain. Pain Pract. 2011; 11(3): 290–296. PubMed Abstract \| Publisher Full Text\n\nSusheela AT, Packiasabapathy S, Gasangwa DV, et al.: Dataset 1 in: The use of dexmedetomidine and intravenous acetaminophen for the prevention of postoperative delirium in cardiac surgery patients over 60 years of age: a pilot study. F1000Research. 2017. Data Source" }	[ { "id": "27423", "date": "09 Nov 2017", "name": "Nicolai Goettel", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this single center randomized controlled trial (RCT), Susheela and coworkers investigate the use of dexmedetomidine and intravenous acetaminophen for the prevention of postoperative delirium in elderly patients undergoing cardiac surgery. This pilot study primarily aimed to assess the feasibility of a larger RCT with identical interventions.\nAs a pilot project, the study is methodically sound. Overall, the manuscript is well-written and pleasant to read. The authors applied CONSORT criteria which is a definite plus.\nPostoperative delirium has specific features that may be related to surgery (and anesthesia) and has a probable neuroinflammatory component. I therefore do not agree with your statement that 30-40% of delirium cases could be prevented, at least not in your sample of patients undergoing cardiac surgery. So far, multicomponent interventions to prevent delirium have only been proven efficacious in a general population of elderly hospitalized patients.1\nPlease also discuss the opioid-sparing effects of intraoperative dexmedetomidine. Next to the missing interaction with GABA receptors, this might be an additional mechanism for delirium prevention when using dexmedetomidine.\nPlease discuss the reasons to exclude patients with preexisting cognitive impairment from your study. Preoperative cognitive impairment was found to be one of the most important risk factors for postoperative delirium (odds ratio ranged from 6.3 (95% CI 2.9 to 13.7) up to 11.5 (95% CI 6.1 to 20.1)).2 By excluding these patients (also in the follow-up RCT), you likely decrease the number of patients affected by the primary outcome measure (postoperative delirium) in your study. It would be interesting to investigate the effects of dexmedetomidine and intravenous acetaminophen in these high-risk patients.\nI find it difficult to blind the intravenous administration of propofol and dexmedetomidine. At best, you may try to conceal the study drug from patient, surgeon, ICU caregivers, and investigators by using opaque syringes and lines. Was study drug administration really double-blind?\nWas there any kind of standardization in anesthesiologic management? If yes, state drugs and dosage.\nAt what frequency did you assess patients for postoperative delirium in the ICU/on the ward? Who assessed the patients?\nI wish the investigators of this pilot project the best of luck with the larger ongoing trial!\n\nReferences\nInouye SK, Bogardus ST Jr, Charpentier PA, et al. A multicomponent intervention to prevent delirium in hospitalized older patients. N Engl J Med 1999; 340: 669–76. National Institute for Health and Care Excellence. NCGC National Clinical Guideline Centre. Delirium: diagnosis, prevention and management. http://www.nice.org.uk/guidance/cg103/evidence/full-guideline-134653069.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3179", "date": "10 Nov 2017", "name": "Ammu Susheela", "role": "Author Response", "response": "Dear Nicolai Goettel, We sincerely thank you for the opportunity to address the comments of our manuscript. We have thoroughly reviewed the comments. In this letter, we address, point-by-point, all the recommendations. We thank you for the review. ResponseIn this single-center randomized controlled trial (RCT), Susheela and coworkers investigate the use of dexmedetomidine and intravenous acetaminophen for the prevention of postoperative delirium in elderly patients undergoing cardiac surgery. This pilot study primarily aimed to assess the feasibility of a larger RCT with identical interventions. As a pilot project, the study is methodically sound. Overall, the manuscript is well-written and pleasant to read. The authors applied CONSORT criteria which is a definite plus. Thank you Postoperative delirium has specific features that may be related to surgery (and anesthesia) and has a probable neuroinflammatory component. I, therefore, do not agree with your statement that 30-40% of delirium cases could be prevented, at least not in your sample of patients undergoing cardiac surgery. So far, multicomponent interventions to prevent delirium have only been proven efficacious in a general population of elderly hospitalized patients.1 Thank you. These estimates are from the pilot study result. The ongoing study will inform us better about the effect size. Please also discuss the opioid-sparing effects of intraoperative dexmedetomidine. Next, to the missing interaction with GABA receptors, this might be an additional mechanism for delirium prevention when using dexmedetomidine. Thank you. Agreed. It is possible that dexmedetomidine can reduce the analgesic requirements. The ongoing study will inform us of this effect size as well due to the crossover design. Please discuss the reasons to exclude patients with preexisting cognitive impairment from your study. Preoperative cognitive impairment was found to be one of the most important risk factors for postoperative delirium (odds ratio ranged from 6.3 (95% CI 2.9 to 13.7) up to 11.5 (95% CI 6.1 to 20.1)).2 By excluding these patients (also in the follow-up RCT), you likely decrease the number of patients affected by the primary outcome measure (postoperative delirium) in your study. It would be interesting to investigate the effects of dexmedetomidine and intravenous acetaminophen in these high-risk patients.Thank you. As this is the first study of this size, we would like to get a homogenous group examined first and definitely the next step is to do the patients with preexisting cognitive dysfunction. I find it difficult to blind the intravenous administration of propofol and dexmedetomidine. At best, you may try to conceal the study drug from patient, surgeon, ICU caregivers, and investigators by using opaque syringes and lines. Was study drug administration really double-blind? Thank you. We only double-blinded the Acetaminophen arms. Was there any kind of standardization in anesthesiologic management? If yes, state drugs and dosage. Cardiac surgery is the most protocolized intraoperative setting. Patients were induced with 500mcg of IV fentanyl, 50-100mg of Propofol, and endotracheal intubation was facilitated with 1mg/kg intravenous rocuronium. Anesthesia was maintained with isoflurane (0.6-1.0 MAC) in 100% oxygen with intermittent fentanyl up to a maximum of additional 500 mcg of fentanyl and intermittent rocuronium. All patients were taken intubated to the ICU At what frequency did you assess patients for postoperative delirium in the ICU/on the ward? Who assessed the patients? Assistants trained from Edward Marcantonio’s lab (with initial half a day training session, witnessing the assessments for a few patients and also calibration with gold standard assessor). Postoperative delirium was assessed by a trained study team member with the help of cognitive assessments such as MMSE, CAM-ICU, and CAM starting from the day after the surgery. If cognitive assessments are negative for 3 consecutive days (i.e days 5, 6 & 7), assessments were completed every other day until the date of discharge. I wish the investigators of this pilot project the best of luck with the larger ongoing trial! Thank you" } ] }, { "id": "27502", "date": "27 Nov 2017", "name": "Michael Avidan", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting and important feasibility study, which seeks to determine whether it is possible to conduct a (large) study evaluating various combinations of sedation/analgesia post cardiac surgery, with the intention of preventing postoperative delirium. In general, with a very small feasibility study, the investigators demonstrate that conducting such a study might be possible. However this study has important constraints.\nThe investigators state, “The mean duration of delirium was 0-1 days.” This appears to be a range.\n\nThe investigators conclude, “The feasibility of performing a large-scale project is ascertained by the study.” I would rather argue that the feasibility of performing a project is ascertained. Whether or not a large-scale project is feasible is not necessarily established.\n\nContrary to the investigators, I do not believe that a 12 patient study with four groups supports the inference “that IVA may have a role in reducing the incidence of delirium.” This is over-interpretive of very limited data.\n\nI do not agree that using the CAM is a major limitation in delirium research (as the investigators suggest that it might lack sensitivity). I do agree that this might apply to the CAM-ICU.\n\nMore efficient tools might not necessarily be more sensitive.\n\nI am not convinced that a 12 patient pilot trial will produce sufficiently precise estimates to power a large RCT appropriately. Although the absolute difference between proportions (delirium incidence) was 50% (67% minus 17%), the 95% confidence interval for this difference is -13% to 82%. Thus, it is not clear based on this small study what value should be used (estimated difference in delirium incidence between the groups [acetaminiphen and placebo]) for the sample size calculation.\n\nFrom the registration site (for NCT02546765), it appears that 120 patients will be included in the (large) trial. This number is likely to be much too small to answer the research question with sufficient precision.\n\nThe reasons for the exclusion criteria are not clear.\n\nWith the modified factorial design, was interaction between interventions considered? Specifically, both dexmedetomidine and acetaminophen might have an impact on the outcome of interest (delirium).\n\nThe (secondary) results of the study were as follows: “The Propofol+Acetaminophen group had no occurrence of delirium. Interestingly, only 17% (1/6) of the subjects who received IVA were diagnosed with delirium compared to 67% (4/6) in the group who did not receive IVA.” The investigators should be cautious about over-interpreting these findings, or using these estimates to justify sample size for a large trial. The problem with this would be that 67% is a very high incidence, and is not likely to reflect the incidence with current standard of care (i.e., propofol or dexmedetomidine sedation, without intravenous acetaminophen). Similarly, the incidence of 17% is actually likely to be pretty close to the current delirium incidence in the type of patients who were enrolled to this study (but who receive standard of care).\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3272", "date": "14 Dec 2017", "name": "Ammu Susheela", "role": "Author Response", "response": "Dear Dr. Micheal Avidan,Thank you for reviewing the paper. We have thoroughly reviewed the comments. We are addressing below, point-by-point all the suggestions. The investigators state, “The mean duration of delirium was 0-1 days.” This appears to be a range.Thank you. Yes. We reported the mean duration as a range. The investigators conclude, “The feasibility of performing a large-scale project is ascertained by the study.” I would rather argue that the feasibility of performing a project is ascertained. Whether or not a large-scale project is feasible is not necessarily established.Thank you. We agree that only the feasibility of performing a project is ascertained. Contrary to the investigators, I do not believe that a 12 patient study with four groups supports the inference “that IVA may have a role in reducing the incidence of delirium.” This is over-interpretive of very limited data.Thank you. We agree this is a pre-pilot study indeed. It was a feasibility project and we certainly do not want to overinterpret. Thus, we wrote “IVA may be beneficial in reducing the incidence of delirium following cardiac surgery. ” Since this is a feasibility study we agree that a larger study is needed to draw a concrete conclusion. I do not agree that using the CAM is a major limitation in delirium research (as the investigators suggest that it might lack sensitivity). I do agree that this might apply to the CAM-ICU.Thank you. Agreed. More efficient tools might not necessarily be more sensitive.Thank you. Agreed. I am not convinced that a 12 patient pilot trial will produce sufficiently precise estimates to power a large RCT appropriately. Although the absolute difference between proportions (delirium incidence) was 50% (67% minus 17%), the 95% confidence interval for this difference is -13% to 82%. Thus, it is not clear based on this small study what value should be used (estimated difference in delirium incidence between the groups [acetaminiphen and placebo]) for the sample size calculation.Thank you. We planned this pilot study as a means to assess the feasibility of performing a large-scale project. However, there is no other preexisting data to power the next larger trial. Since this pilot study showed a 66% reduction in the incidence of delirium, for the power calculation, a 50% reduction in the delirium incidence was used. Each group needed 56 patients (alpha=0.05 and 1-Beta=0.80). From the registration site (for NCT02546765), it appears that 120 patients will be included in the (large) trial. This number is likely to be much too small to answer the research question with sufficient precision.Thank you. A multicenter trial is definitely required to answer with precision. However, it will be a major leap to go from a pilot study to a multicenter trial. Thus, we chose to do the second step of testing in a larger sample size with what is known so far. Depending on what this study shows, the obvious next step will be a larger multicenter trial. The reasons for the exclusion criteria are not clear.Thank you. We chose a homogenous set of patients to determine the drug efficacy. We wanted to avoid patients who might not get extubated in a reasonable amount of time (hence the EF, aortic surgery exclusion, etc). As we chose to give Q6H IV acetaminophen, we wanted to avoid any clinical situation that can potentially set up patients for drug toxicity etc. Preexisting cognitive dysfunction, etc. could muddle the picture in a 120 patient study. In a larger multicenter trial if and when launched, the heterogeneity can be accounted for and thus need not necessarily be an exclusion criteria. With the modified factorial design, was interaction between interventions considered? Specifically, both dexmedetomidine and acetaminophen might have an impact on the outcome of interest (delirium).Thank you. Yes, these interactions will be analyzed in the ongoing trial The (secondary) results of the study were as follows: “The Propofol+Acetaminophen group had no occurrence of delirium. Interestingly, only 17% (1/6) of the subjects who received IVA were diagnosed with delirium compared to 67% (4/6) in the group who did not receive IVA.” The investigators should be cautious about over-interpreting these findings, or using these estimates to justify sample size for a large trial. The problem with this would be that 67% is a very high incidence, and is not likely to reflect the incidence with current standard of care (i.e., propofol or dexmedetomidine sedation, without intravenous acetaminophen). Similarly, the incidence of 17% is actually likely to be pretty close to the current delirium incidence in the type of patients who were enrolled to this study (but who receive standard of care).Thank you for the caution. We agree with all the comments." } ] } ]	1	https://f1000research.com/articles/6-1842
https://f1000research.com/articles/6-2164/v1	21 Dec 17	{ "type": "Software Tool Article", "title": "Abstract Sifter: a comprehensive front-end system to PubMed", "authors": [ "Nancy Baker", "Thomas Knudsen", "Antony J. Williams", "Thomas Knudsen", "Antony J. Williams" ], "abstract": "The Abstract Sifter is a Microsoft Excel based application that enhances existing search capabilities of PubMed. The Abstract Sifter assists researchers to search effectively, triage results, and keep track of articles of interest. The tool implements an innovative “sifter” functionality for relevance ranking, giving the researcher a way to find articles of interest quickly. The tool also gives researchers a view of the literature landscape for a set of entities such as chemicals or genes. The Abstract Sifter is available as a Microsoft Excel macro-enabled workbook application.", "keywords": [ "PubMed", "document retrieval", "Microsoft Excel", "Visual Basic for Applications", "chemistry", "toxicology", "biology" ], "content": "Introduction\n\nScientists in the biological and medical domains can spend considerable time searching for relevant articles in PubMed, and tools that make the searching more effective will save time and resources (Khare et al., 2014). Here, we present a tool, the Abstract Sifter, built to improve efficiency in searching PubMed. Specifically, this tool was designed with the following objectives: 1) To make it quicker and easier to find relevant articles in PubMed; 2) To visualize the “literature landscape”, which can help focus on key relevant articles; 3) To make it easier to evaluate and take notes on abstracts; and 4) To facilitate collaboration on literature tasks.\n\n\nMethods\n\nThe Abstract Sifter application is a Microsoft Excel macro-enabled workbook that has been tested in Excel 2013 and 2016 on the Windows platform. Visual Basic for Applications (VBA) was used to develop the features that go beyond native Excel functionality. For the retrieval of PubMed query results, Entrez Programming Utilities (E-utilities) (Sayers, 2016) are called from VBA. These utilities were developed by the National Center for Biotechnology Information (NCBI) to allow software developers to query PubMed and other NCBI databases and retrieve the results for incorporation into local applications (2017). Through implementation as an Excel workbook, the Abstract Sifter can easily be shared with collaborators.\n\n\nUse case\n\nThe Abstract Sifter application workbook contains seven sheets: ReadMe, Main, Abstract, Notes, Log, and Landscape, and SampleQueries. The Main sheet is where the basic functions operate, including the novel functionality called “sifting”. To start, the end-user clicks on the Query PubMed button at the top of the screen and enters any PubMed query of interest. For the example in Figure 1a, the end-user has entered the simple query “chlorpyrifos”. However, these queries can be more complex. In fact, any query run in PubMed can be executed in the Sifter. When the query entry is finished, the user then clicks on Submit and the query is sent to the NCBI PubMed E-utility. The first response returned by the E-utility is the number of articles found that satisfy the query. The citations are downloaded from PubMed by the Abstract Sifter, parsed by pattern matching algorithms coded in VBA. All citations are thus parsed for title, abstract, authors, publication year, journal, and PubMed identifier, and the data is inserted into rows in the Main sheet. Every new search will clear results from the previous query. For performance purposes, if the number of articles exceeds 5,000, the query will not be run and the user is encouraged to re-word the query to return fewer records.\n\n1a. Abstract Sifter Main sheet and 1b. Abstract sheet view.\n\nThe results of the query stored in the Main sheet can be browsed like any other data in a spreadsheet. The sifter feature provides a novel and effective way to narrow search results with large number of citations to find articles of interest. For example, the query for “chlorpyrifos” returned over 4,000 PubMed citations. If a researcher is looking for neurological effects in studies where rats were dosed with chlorpyrifos, the researcher could type the term “mg/kg” in the spreadsheet cell B3, “rat” in C3, and “brain” in D3 (Figure 1a inset). The Abstract Sifter returns the number of occurrences of each term found in the title and abstract combined. The Main sheet’s citations can be sorted by these counts. Sifting by entering terms and sorting can be repeated. Similarly, new PubMed queries can be run, altered, and rerun. Double-clicking on any cell in the row (except the cell containing the PMID) takes the end-user to the Abstract sheet where the title and abstract of that citation are shown (Figure 1b). The sifter terms are highlighted by giving each the color of the term on the Main sheet. Together, these query and sifting capabilities provide a powerful search tool.\n\nThe Abstract Sifter also incorporates functionality to allow the end-user to take notes on citations. On the Abstract sheet, for instance, the user can click on the button Add Note. A form appears that provides the opportunity to add short notes (tags) or long notes or to specify one of three categories (yes, no, or maybe) (Figure 2a). How these values are used is a decision of the end-user. When the user clicks on OK, a row is inserted into the Notes sheet with the citation information along with the notes. Alternatively, the end-user can take notes on more than one article at a time from the Main sheet. To do this, the end-user selects multiple rows of interest and then clicks on Take Group Notes. Each of the selected citations will be inserted into the Notes sheet with the entered notes and tags (Figure 2b).\n\n2a. Taking Group notes. 2b. Viewing Notes sheet. 2c. Highlighting noted citations.\n\nOften, after entering a number of notes, the user will forget which citations have been read and evaluated and for which notes have been taken. By clicking on the Highlight Noted PMIDs button either on the Main or Notes sheet, the PubMed identifier (PMID) on the Main sheet will be set to the color specified in the Note form (Figure 2c). Using the built-in Excel filtering feature, the color can be selected or sorted on to view Noted citations.\n\nThe Notes sheet itself can be viewed and edited and rows can be deleted. Entries on the Notes sheet can be exported to PubMed where they can then be downloaded in a number of different formats, including a format for direct import into citation management software. The button to export to a citation manager via PubMed is labelled Get references and appears on the Notes sheet.\n\nAnother unique feature of the Abstract Sifter is the Landscape sheet functionality. The Landscape sheet is an alternative to the Main sheet as an entry point, and provides the end-user a visualization of literature for a set of chemicals. To use this functionality, the end-user enters chemical name queries in Column C of the Landscape sheet after Row 4. An example in which the end-user has entered seven chemical queries is shown in Figure 3. The chemical queries can be extended with CAS registry numbers or synonyms. Next, the end-user enters subject matter query text in Row 3, Columns E and higher. In the example depicted in Figure 3, the end-user has entered several subject matter queries, starting with “neoplasms OR cancer”.\n\nThe end-user can then select cells in the intersection region (E5 through J11 in Figure 3) and click on the button Update Article Counts. This action causes the tool to iterate through the cells, build a query using the chemical terms in column C appended to the effect terms in Row 3, and then send the query to PubMed for execution. The counts of the articles satisfying each query are returned from PubMed and are inserted into the corresponding cells. To see the PubMed records, the user double-clicks on a cell in the intersection region. This action starts the PubMed query process and sends the results to the Main page for sifting. More chemicals, entries and/or and additional queries can be added by the user on this Landscape sheet. Buttons are available on the Landscape sheet to help with formatting results, such as applying heat-map coloring to the article counts. The SampleQueries sheet has some text that can be used as a starting point for Landscape queries. To use, the end-user selects rows and clicks on the Send Queries to Landscape button to have the queries appended to Row 3 on the Landscape page.\n\nThe Log sheet contains a row for each query run. The query text is inserted into the row along with date and time information and the number of records returned. Queries can be easily rerun by double-clicking on the query text in column C.\n\n\nDiscussion\n\nThe Abstract Sifter can facilitate many PubMed literature tasks by enabling rapid identification, triage, and tracking of relevant articles. The literature landscape viewing and navigating capabilities give researchers distinctive insight into characteristics of a literature corpus.\n\n\nSoftware availability\n\nThe Abstract Sifter Excel workbook and user documentation are available at: https://github.com/USEPA/CompTox-Chemistry-Dashboard-Abstract-Sifter/\n\nArchived source code as at the time of publication: http://dx.doi.org/10.5281/zenodo.1040961 (nancycolebaker, 2017)\n\nLicense: CC0 1.0 Public Domain Dedication.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nResearch in this publication was supported by the U.S. Environmental Protection Agency. The views expressed in this paper are those of the authors and do not necessarily represent the views or policies of the U.S. Environmental Protection Agency.\n\n\nReferences\n\nKhare R, Leaman R, Lu Z: Accessing biomedical literature in the current information landscape. Methods Mol Biol. 2014; 1159: 11–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nnancycolebaker: nancycolebaker/LitInfo: PubMed Abstract Sifter public release v.1. (Version v1). Zenodo. 2017. Data Source\n\nNCBI Resource Coordinators: Database Resources of the National Center for Biotechnology Information. Nucleic Acids Res. 2017; 45(D1): D12–D17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSayers E: A General Introduction to the E-utilities. 2016; Retrieved 2016. Reference Source" }	[ { "id": "29810", "date": "15 Jan 2018", "name": "Pauliina Damdimopoulou", "expertise": [ "Reviewer Expertise Molecular biology", "environmental medicine", "endocrine disrupters", "hormone signalling", "female fertility" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a Microsoft Excel workbook based tool for rapid PubMed literature searches where the results can be easily sifted and annotated. With the ever-growing body of literature, tools for finding and sorting literature become increasingly more important. I am not a software developer myself, and cannot therefore evaluate the technical aspects of the tool (codes, macros, algorithms…). However, I am a keen end-user of tools such as Abstract Sifter, because my work is highly interdisciplinary and I often need to rapidly find information about unfamiliar topics, and can therefore estimate the user friendliness and usefulness of this new tool.\nA clear benefit of Abstract Sifter is its Excel format. Most researchers are familiar with Excel and therefore starting to work with Abstract Sifter does not require any extra effort to understand the interphase. It is convenient that different searches can be saved and shared with colleagues like any file. I can also vision sharing a Sifter file through a cloud service and simultaneously annotating and going through literature for example for a review or grant application.\nI have carefully read through the article (last updated 02 JAN 2018) and the User Guide (version 1.0) as well as tried the different functions of Abstract Sifter (v1), and find only minor issues to comment on. I think the work is of acceptable standard to be approved.\nMinor comments\nBuilding queries in the Sifter. It would be helpful to have an ”advanced query builder” option available. When I was trying different queries, I ended up building the more complicated ones using the PubMed advanced search builder and copy pasting the query line to Sifter.\n\nDoes the Sifter function take article keywords and authors into consideration in the counts?\n\nPage 3: ”Highlight Noted PMIDs” should be ”Highlight Noted”.\n\nThe ”For HAWC” button on the Notes sheet is not explained in the article (please also see comment 7).\n\nThe Helpful Tips in the user guide are very useful! One to add could be how to retrieve selected references from PubMed with the ”Get references” button. For example, if I have gone through and annotated the 4,000 chlorpyrifos citations and only want to retrieve the 150 I’ve marked ”yes” (green) in the Notes sheet, how should I do that?\n\nThe Landscape function is an exciting way to quickly get an idea of how well certain topics have been covered in the literature. What is Column D ”Link” in the Landscape sheet? What is the relationship between Rows 3 and 4? Is row 4 just user defined short names for row 3 queries, and not taken into account in the PubMed based search for citations? This could be clarified in the article text. The Landscape heatmap colours could also be explained too together with their cut-offs (I assume it’s based on % instead of absolute counts?). Should 0 labelled with white or blue?\n\nOn the ReadMe sheet in the Shifter file, ”Chemical sheet” is mentioned, although the Sifter does not have one with that name. Do you maybe mean Landscape sheet? ReadMe page also gives a link to a HAWC video, although HAWC is not explained in the article (see also comment 4).\n\nUser Manual page 10: ”…short version of the chemical name is in Column A” should be ”…Column B”. Column A is called ”DSSTOX link to Dashboard” in the file.\n\nI wonder, if I were to use this tool with a colleague to select citations for a review, for example, is there a way to add ”comments” to the notes? For example, if I have classified a citation as ”yes” but my colleague does not agree, how could that be easily marked into the Notes page?\n\nDoes the Shifter work both in Mac and PC environment? What about Open Office?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "30158", "date": "06 Feb 2018", "name": "Qingliang (Leon) Li", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors reported an Excel front-end for PubMed search by using NCBI E-Utilities. The tool supports searching literature in PubMed, importing search results into excel, adding notes into individual literature etc. Here are some minor issues that the authors may consider to improve the usabilities of the tool:\n\nCurrently, the tool has a limit of 5000 results for a search. This makes sense when considering excel performance, however, it will improve the usability if the tool can support pagination and/or have the user to select which literature to import.\n\nThe term search/count feature in the main sheet can be improved by restricting the \"searched term\" to be a \"real term\". For example, \"generation\" that has nothing related to \"rat\" should not be highlighted as a 'rat term'.\n\nIn the landscape view, the authors may consider linking chemical name to chemical databases, such as PubChem, to view chemical structures.\n\nThe authors may consider connecting this tool with \"My NCBI\" (https://www.ncbi.nlm.nih.gov/account/?back_url=https%3A%2F%2Fwww.ncbi.nlm.nih.gov%2Fmyncbi%2F)\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2164
https://f1000research.com/articles/6-2163/v1	21 Dec 17	{ "type": "Research Note", "title": "Whole exome sequencing identifies a novel homozygous frameshift mutation in the ASPM gene, which causes microcephaly 5, primary, autosomal recessive", "authors": [ "Desaraju Suresh Bhargav", "N. Sreedevi", "N. Swapna", "Soumya Vivek", "Srinivas Kovvali", "Desaraju Suresh Bhargav", "N. Sreedevi", "N. Swapna", "Soumya Vivek" ], "abstract": "Microcephaly is a genetically heterogeneous disorder and is one of the frequently notable conditions in paediatric neuropathology which exists either as a single entity or in association with other co-morbidities. More than a single gene is implicated in true microcephaly and the list is growing with the recent advancements in sequencing technologies. Using massive parallel sequencing, we identified a novel frame shift insertion in the abnormal spindle-like microcephaly-associated protein gene in a client with true autosomal recessive primary microcephaly. Exome sequencing in the present case helped in identifying the true cause behind the disease, which helps in the premarital counselling for the sibling to avoid future recurrence of the disorder in the family.", "keywords": [ "Microcephaly", "ASPM", "Exome Sequencing", "MCPH5", "MCPH" ], "content": "Introduction\n\nMicrocephaly with no other anomalies in the brain structure is termed as true microcephaly or autosomal recessive primary microcephaly (MCPH), where the pathology of brain is generally congenital and static with mild to moderate intellectual disability (ID) (http://www.orpha.net/consor/cgi-bin/OC_Exp.php?Expert=2512). Microcephaly is seen in numerous syndromes1 and even in true microcephaly, there is possibility of more than one gene implicated2,3, thus screening for a single gene may not be very fruitful in this population. Recently, whole exome sequencing (WES) has emerged as a potential approach to delineate the molecular pathology in the microcephaly population with ID1. Using WES, we report a de novo frame shift (insertion) mutation in the calponin-homology domain of ASPM (Abnormal Spindle-Like, Microcephaly-Associated) gene which is a candidate for MCPH5 (Microcephaly 5, primary, autosomal recessive) in a client with true microcephaly.\n\n\nReport\n\nThe client sequenced was under the research project cerebral palsy and spectrum conditions (seizures, mental retardation, microcephaly and other neurodevelopmental disorders), which aims to find disease causing mutations in a sample of 100 clients recruited from our Motor Speech Disorders Clinic at Department of Clinical Services, All India Institute of Speech and Hearing. This is a research project and hence ethical clearance was obtained, reference was mentioned in Methods. The client is a 15 year old female born out of a consanguineous union (parents were first cousins) diagnosed with microcephaly (occipitofrontal head circumference was 40 centimetres) and developmental delay. The mother had a history of one miscarriage at the third month of gestation and a still birth (female) at the eighth month (Figure 1A). An ultra sound scan of the foetus (client) at the eight month of pregnancy revealed delayed development. The client was born at term through normal delivery with no birth trauma. Her younger brother was clinically normal. The client had a squint at birth and had mild ID. At age 12 years, her developmental age was between 66 to 78 months as assessed by the Developmental Screening Test (DST)4. Her Receptive Language Age (RLA) and Expressive Language Age (ELA) was 18–20 months as revealed by Receptive Expressive Emergent Language Scale (REELS)5. She had a vocabulary of around 50 words and was able to comprehend commands, would recognize family members and common objects. She expressed herself through single word utterances, gestures and pointing.\n\n(A) The pedigree of the family; (B) workflow of the variant prioritization.\n\nThe study was approved by the Institutional Ethical Body, All India Institute of Speech and Hearing [Ethical clearance reference number: SH/CDN/ARF-40/2016-17]. After obtaining written informed consent from the parents of the client, 5 ml of blood was collected from all family members (mother, father and brother) into EDTA coated vacutainers and DNA was isolated using Pure Link Genomic DNA Isolation Kit (Thermo Fisher Scientific), as per the manufacturer’s instructions. Approximately 100ng of genomic DNA was used to construct Exome libraries using Ion Ampliseq Exome RDY Panel (Thermo Fisher Scientific), as per the manufacturer’s protocol and these were quantified using High Sensitivity genomic DNA Assay on Qubit 3.0 (Thermo Fisher Scientific). Approximately 25 Pico moles of the library was used with the Ion Chef Instrument (Thermo Fisher Scientific) for template generation followed by, enrichment of templated ion sphere particles. Sequencing was performed using Hi-Q chemistry on Ion Proton system (Thermo Fisher Scientific) at our facility. Two samples were sequenced per chip and run generated 10.98 GB. The sample in question yielded 28,105,510 reads with 185 mean base pair length.\n\n\nResults\n\nQC filtered reads were aligned to reference genome GRCh37/hg19. Of the 28.1 million reads, 99.27% reads were on target with a mean genomic coverage of 91.83%. Mean depth was 81.32x with 89.87% mean uniformity. Variants were called from raw data using inbuilt variant caller plug-in present in Torrent suite (Version 5.2.2). Ion Reporter (version 5.4) annotated 38,085 variants from 13,013 genes to hg19 from the VCF file generated by variant caller plug-in. Variant prioritization was performed as shown in Figure 1B.\n\nFrom the pedigree chart, we assumed autosomal recessive inheritance and filtered out heterozygous variants. This resulted in 75 homozygous variants from 67 genes. We found a novel frame shift insertion in exon 13 of the ASPM gene [(NM_018136.4), c.3384_3385 Insertion T], which induces a termination codon (p.Lys1129Ter) leading to non functional ASPM protein. Insertion was verified by Sanger sequencing using BDTv3 on 3500 Genetic Analyzer (Thermo Fisher Scientific). Homozygous insertion was confirmed in the client. Both parents and unaffected sibling were found to be heterozygous carriers (Figure 1A).\n\n\nDiscussion\n\nASPM protein determines cerebral cortical size. During initial stages of corticogenesis, the ASPM protein is essential in facilitating the proliferation of neural progenitors6. This process determines the cerebral cortical volume7 which has tripled over the last ~ 2 million years, leading to exceptionally big brain in humans compared to their primate counterparts8. This increase in the human brain size is believed to be one contributing factor for the emergence of higher cognitive function and language ability that are restricted to humans8. So far, 17 genes have been reported in which, mutations lead to the development of MCPH3,4,9–26. The phenotype(s) arising from pathogenic variants in these 17 genes are each named from MCPH 1 – MCPH 17 (there are 17 genes identified so far that cause autosomal recessive primary microcephaly, MCPH arising from these 17 genes are termed from MCPH 1 to MCPH 17) and the majority of the genetic load in MCPH is contributed by the ASPM gene, making MPCH5 the most prevalent of all the types of MCPH. Frame shift and protein truncating mutations in ASPM cause MCPH5 and these mutations are restricted to be seen in homozygous state only in the MCPH5 population27,28 (i.e., heterozygous mutations does not have any effect and only homozygous mutations will cause the MCPH).\n\n\nConclusion\n\nThe novel insertion mutation found in the ASPM gene in the present study segregated with the phenotype in the family, establishes the role of the novel frame shift mutation identifiedin the development of MCPH5 in the case studied. Candidate gene study by Sanger sequencing is time consuming and not economical when compared to WES. Given its higher diagnostic yield as evident by published studies on neurodevelopmental disorders2 and also from the present work, we support the findings reported by Rump et al. (2016) which states that WES in microcephaly population will end unnecessary further evaluations and aid in early appropriate interventions2.\n\n\nConsent\n\nWritten informed consent to carry out the study and for the publication of the client’s and client’s sibling’s clinical details were obtained from the parents. Clinical details were obtained from the parents of the client.\n\n\nData availability\n\nSequence data for the insertion mutation (client sample) was deposited in Genbank under accession number MG063723.", "appendix": "Author contributions\n\n\n\nSBD performed Exome Sequencing and Sanger sequencing, NS, NS and SK conceived the study, SV collected the data and its curation, SBD, SK performed data analysis and prepared manuscript, NS, NS supervised the work and corrected the draft, all the authors have read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nGrants to carry out the present project were provided by All India Institute of Speech and Hearing, an autonomous Institute under Ministry of Health and Family Welfare, Government of India, India.\n\n\nAcknowledgements\n\nThe authors thank the participants for their cooperation. Our sincere thanks to Dr. S.R.Savithri, Director, All India Institute of Speech and Hearing, Mysore for permitting us to undertake the work.\n\n\nReferences\n\nRump P, Jazayeri O, van Dijk-Bos KK, et al.: Whole-exome sequencing is a powerful approach for establishing the etiological diagnosis in patients with intellectual disability and microcephaly. BMC Med Genomics. 2016; 9: 7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAbdel-Hamid MS, Ismail MF, Darwish HA, et al.: Molecular and Phenotypic Spectrum of ASPM-Related Primary Microcephaly: Identification of Eight Novel Mutations. Am J Med Genet Part A. 2016; 170(8): 2133–40. PubMed Abstract \| Publisher Full Text\n\nHashmi JA, Al-Harbi KM, Ramzan K, et al.: A novel splice-site mutation in the ASPM gene underlies autosomal recessive primary microcephaly. Ann Saudi Med. 2016; 36(6): 391–396. PubMed Abstract \| Publisher Full Text\n\nBharath Raj J: DST Manual +Know your child’s intelligence and how to improve it. Sri Meera Printers: Mysore, 1983.\n\nBzoch K, League R: Receptive-Expressive Emergent Language (REEL) scale. Austin, TX:PRO-ED, 1971.\n\nBuchman JJ, Durak O, Tsai LH: ASPM regulates Wnt signaling pathway activity in the developing brain. Genes Dev. 2011; 25(18): 1909–1914. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKouprina N, Pavlicek A, Mochida GH, et al.: Accelerated Evolution of the ASPM Gene Controlling Brain Size Begins Prior to Human Brain Expansion. PLoS Biol. 2004; 2(5): E126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J: Evolution of the Human ASPM Gene, a Major Determinant of Brain Size. Genetics. 2003; 165(4): 2063–2070. PubMed Abstract \| Free Full Text\n\nJackson AP, Eastwood H, Bell SM, et al.: Identification of microcephalin, a protein implicated in determining the size of the human brain. Am J Hum Genet. 2002; 71(1): 136–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRoberts E, Jackson AP, Carradice AC, et al.: The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1-13.2. Europ J Hum Genet. 1999; 7(7): 815–820. PubMed Abstract \| Publisher Full Text\n\nMoynihan L, Jackson AP, Roberts E, et al.: A third novel locus for primary autosomal recessive microcephaly maps to chromosome 9q34. Am J Hum Genet. 2000; 66(2): 724–727. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJamieson CR, Govaerts C, Abramowicz MJ: Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15. Am J Hum Genet. 1999; 65(5): 1465–1469. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPattison L, Crow YJ, Deeble VJ, et al.: A fifth locus for primary autosomal recessive microcephaly maps to chromosome 1q31. Am J Hum Genet. 2000; 67(6): 1578–1580. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLeal GF, Roberts E, Silva EO, et al.: A novel locus for autosomal recessive primary microcephaly (MCPH6) maps to 13q12.2. J Med Genet. 2003; 40(7): 540–542. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKumar A, Girimaji SC, Duvvari MR, et al.: Mutations in STIL, encoding a pericentriolar and centrosomal protein, cause primary microcephaly. Am J Hum Genet. 2009; 84(2): 286–290. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHussain MS, Baig SM, Neumann S, et al.: A truncating mutation of CEP135 causes primary microcephaly and disturbed centrosomal function. Am J Hum Genet. 2012; 90(5): 871–878. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJamieson CR, Govaerts C, Abramowicz MJ: Primary autosomal recessive microcephaly: homozygosity mapping of MCPH4 to chromosome 15. Am J Hum Genet. 1999; 65(5): 1465–1469. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYang YJ, Baltus AE, Mathew RS, et al.: Microcephaly gene links trithorax and REST/NRSF to control neural stem cell proliferation and differentiation. Cell. 2012; 151(5): 1097–1112. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAwad S, Al-Dosari MS, Al-Yacoub N, et al.: Mutation in PHC1 implicates chromatin remodeling in primary microcephaly pathogenesis. Hum Mol Genet. 2013; 22(11): 2200–2213. PubMed Abstract \| Publisher Full Text\n\nHussain MS, Baig SM, Neumann S, et al.: CDK6 associates with the centrosome during mitosis and is mutated in a large Pakistani family with primary microcephaly. Hum Molec Genet. 2013; 22(25): 5199–5214. PubMed Abstract \| Publisher Full Text\n\nMirzaa GM, Vitre B, Carpenter G, et al.: Mutations in CENPE define a novel kinetochore-centromeric mechanism for microcephalic primordial dwarfism. Hum Genet. 2014; 133(8): 1023–1039. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKhan MA, Rupp VM, Orpinell M, et al.: A missense mutation in the PISA domain of HsSAS-6 causes autosomal recessive primary microcephaly in a large consanguineous Pakistani family. Hum Molec Genet. 2014; 23(22): 5940–5949. PubMed Abstract \| Publisher Full Text\n\nAlakbarzade V, Hameed A, Quek DQ, et al.: A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome. Nat Genet. 2015; 47(7): 814–817. PubMed Abstract \| Publisher Full Text\n\nGuemez-Gamboa A, Nguyen LN, Yang H, et al.: Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome. Nat Genet. 2015; 47(7): 809–813. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYamamoto S, Jaiswal M, Charng WL, et al.: A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases. Cell. 2014; 159(1): 200–214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBasit S, Al-Harbi KM, Alhijji SA, et al.: CIT, a gene involved in neurogenic cytokinesis, is mutated in human primary microcephaly. Hum Genet. 2016; 135(10): 1199–1207. PubMed Abstract \| Publisher Full Text\n\nBarbelanne M, Tsang WY: Molecular and cellular basis of autosomal recessive primary microcephaly. Biomed Res Int. 2014; 2014: 547986. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNicholas AK, Swanson EA, Cox JJ, et al.: The molecular landscape of ASPM mutations in primary microcephaly. J Med Genet. 2009; 46(4): 249–253. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "29354", "date": "12 Jan 2018", "name": "Shahid Mahmood Baig", "expertise": [ "Reviewer Expertise Monogenic disorders", "neurodevelopmental disorders", "hemoglobinopathies" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this report, a novel frameshift insertion in exon 13 of the ASPM gene in an Indian patient with autosomal recessive primary microcephaly has been reported.\n\nThis finding adds to the spectrum of mutations in ASPM gene using whole exome sequencing (WES) for premarital screening and prenatal diagnosis to prevent affected births in at risk pregnancies. This report justifies the use of WES in delineating MCPH as compared to candidate gene approach. It is a descent short report, and hence recommended for indexing in its present form.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "29314", "date": "15 Jan 2018", "name": "Sulman Basit", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Bhargav and colleagues describes a novel homozygous frameshift insertion mutation in a known microcephaly gene in a family segregating autosomal recessive primary microcephaly.\n\nWhole exome sequencing was used to identify a mutation underlying MCPH phenotype.\n\nOverall, the manuscript is written well. Authors have presented the data in a professional way and I have no reservation to approve this submission.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2163
https://f1000research.com/articles/6-2155/v1	20 Dec 17	{ "type": "Research Note", "title": "Vasomotor symptoms monitoring with a commercial activity tracking watch", "authors": [ "Darrell O. Ricke" ], "abstract": "Personal fitness/health tracking devices that include electrodermal activity sensors enable tracking of vasomotor symptoms (hot flashes). Multiple conditions are associated with vasomotor symptoms. This article describes nighttime tracking of vasomotor symptoms for an individual over a two-year period. This volunteer was a participant in a longitudinal study on volunteers wearing physiological monitors. Personal tracking of vasomotor symptoms will provide new insights on the differences between conditions and impacts on individual’s health.", "keywords": [ "Vasomotor symptoms", "hot flashes", "menopause", "galvanic skin response", "GSR", "electrodermal activity", "EDA", "physiological monitoring", "health tracking" ], "content": "Introduction\n\nContinuous tracking of electrodermal activity (EDA), also known as galvanic skin response (GSR), values with commercial fitness devices for individuals with vasomotor symptoms (hot flashes) provides a path forward for future studies with fine resolution monitoring. This can improve upon the current reliance on the use of personal diaries (Regestein et al., 2015). There are multiple conditions associated with vasomotor symptoms including menopause/early menopausal transition (Hale et al., 2014), medications (Quinestrol, tramadol, etc.), chemotherapy and Tamoxifen, hyperthyroidism, infections (Inflammatory Bowel Disease – IBD, etc.), and more. Multiple studies have characterized hot flashes in premenopausal and menopausal women using self reported methods, laboratory polysomnographic recording, and some specially designed devices (Freedman, 2014). However, it is difficult for an individual to track and accurately report nighttime vasomotor symptoms without the aid of a physiological monitoring device (Freedman, 2014). Emerging commercial and custom devices with EDA meters will greatly facilitate the nighttime monitoring of hot flashes for individuals for more informative longitudinal studies of conditions with associated vasomotor symptoms. This report illustrates the potential fine-resolution monitoring of nighttime vasomotor symptoms using commercially available activity-tracking devices with an EDA sensor.\n\n\nMethods\n\nMIT Lincoln Laboratory conducted a longitudinal study on volunteers wearing physiological monitors (June 15 2014 to October 2 2016). The study protocol and written consent form were reviewed and approved by the MIT Committee on the Use of Humans as Experimental Subjects (COUHES). The commercial devices in the study include the Basis B1 watch and Basis Peak watch monitors for tracking heart rate, sleep with predicted sleep phases (light, deep, and REM – rapid eye movement), activity, skin temperature, and perspiration (EDA/GSR) without the use of electrodes or gel. The Basis B1 and Peak watches are no longer commercially available, but similar devices with EDA/GSR sensors are available.\n\nVasomotor symptoms started disrupting the sleep of a female volunteer on November 23, 2015, calling attention to their occurrence. After November 23, 2015, the volunteer started personally logging the occurrence of vasomotor symptoms, noting that the recorded EDA signals reflected the logged hot flash intensities and durations.\n\n\nResults\n\nFigure 1 shows data from eight days around this time period. Freedman (Freedman, 1989) identified a good agreement between an increase of 2 μS/cm in 30 s time period with volunteer self-reports for vasomotor symptoms. For November 15th, the EDA median value was 6.8e-4 μS/cm and average value of 4.9e-2 4 μS/cm. For November 16th, the EDA median value was 6.8e-4 μS/cm and average value of 1.9e-2 μS/cm. Across all nights tracked, the EDA median value was 3.7e-4 μS/cm illustrating baseline EDA values for this volunteer. The longitudinal data collected indicates that the volunteer’s vasomotor symptoms may have been occurring as early as June 2014, but went unnoted until higher intensity vasomotor symptoms caused sleep disruptions. A review of all sleep time data indicates an increase in sleep interruption minutes as reported by Basis (52/1957=2.9% for EDA range 10-15 μS/cm and 15/311=4.8% for EDA range 15-25 μS/cm compared to 3823/258119=1.5% for EDA < 1 μS/cm). This is consistent with volunteer observations. Nights like November 23 and 24, 2015 cause sleep disruptions. Figure 2 illustrates over two years of nighttime EDA values while this volunteer was sleeping. Starting in December 2015, the volunteer started self-tracking EDA values and vasomotor symptoms. Daytime peaks were associated with both exercise and vasomotor symptoms. The volunteer reports that daytime vasomotor symptoms and sleep-disrupting vasomotor symptoms were consistent with recorded EDA peaks but they did not record these observations. Nighttime EDA peaks well above baseline values were observed in clusters from June 2014 until November 2016. Note that nights with low EDA values still occur frequently for this volunteer, indicating nights free of vasomotor symptoms. The volunteer did not take hormone or nonhormonal formulations for the treatment of vasomotor symptoms. Note that the volunteer’s Basis B1 watch was replaced with a Basis Peak watch in August 2015.\n\nVertical axis shows EDA values in μS/cm.\n\nHeatmap of each minute of volunteer’s nighttime Basis EDA values tracked with Basis B1 or Basis Peak watch and line plot of nightly maximum EDA values in μS/cm.\n\n\nDiscussion\n\nLongitudinal studies of large numbers of volunteers will provide new foundations for tracking vasomotor symptoms associated with menopause and other conditions. Continuous tracking of EDA values with readily available commercial tracking devices will provide a path forward for future fine-resolution longitudinal studies of conditions associated with vasomotor symptoms. Insights into understanding and treating vasomotor symptoms will be greatly advanced by these longitudinal studies, as the different causes of vasomotor symptoms may vary in intensities and durations. EDA is also reported as a sensitive index of sympathetic nervous system activity (Poh et al., 2010). In addition, commercial devices with EDA meters will be valuable personal monitoring tools to premenopausal and menopausal women and individuals experiencing vasomotor symptoms.\n\n\nData availability\n\nThe nighttime EDA values tracked are available in Ricke, Darrell, 2017, \"GSR nights\", doi: 10.7910/DVN/ONAUUZ, Harvard Dataverse.\n\n\nConsent\n\nWritten informed consent was obtained from the volunteer for the publication of her details.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis material is based upon work supported by the Assistant Secretary of Defense for Research and Engineering under Air Force Contract No. FA8721-05-C-0002 and/or FA8702-15-D-0001. Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the Assistant Secretary of Defense for Research and Engineering. Assistant Secretary of Defense for Research and Engineering has no involvement in this report.\n\nThe funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nI thank Emily Simons for graphics layout support and Paula Collins for careful review of this manuscript.\n\n\nReferences\n\nFreedman RR: Laboratory and ambulatory monitoring of menopausal hot flashes. Psychophysiology. 1989; 26(5): 573–579. PubMed Abstract \| Publisher Full Text\n\nFreedman RR: Menopausal hot flashes: mechanisms, endocrinology, treatment. J Steroid Biochem Mol Biol. 2014; 142: 115–120. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHale GE, Robertson DM, Burger HG: The perimenopausal woman: endocrinology and management. J Steroid Biochem Mol Biol. 2014; 142: 121–131. PubMed Abstract \| Publisher Full Text\n\nPoh MZ, Swenson NC, Picard RW: A wearable sensor for unobtrusive, long-term assessment of electrodermal activity. IEEE Trans Biomed Eng. 2010; 57(5): 1243–1252. PubMed Abstract \| Publisher Full Text\n\nRegestein Q, Friebely J, Schiff I: How self-reported hot flashes may relate to affect, cognitive performance and sleep. Maturitas. 2015; 81(4): 449–455. PubMed Abstract \| Publisher Full Text" }	[ { "id": "29732", "date": "06 Feb 2018", "name": "Nanette Santoro", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe author provides information on a single research study participant who incidentally experienced vasomotor symptoms while testing a physiological monitor in what is presumably a separate study. Changes in electrodermal activity appeared to correlate with self reported night time vasomotor symptoms.\n\nThis case report describes the experience of a single volunteer. There are several details missing that would make it easier to understand how the original study was conducted. The scholarship of the manuscript is also incomplete.\n\nThe parent study in which the participant with vasomotor symptoms was recruited is inadequately described. If already published, a reference to the study would be helpful. If it not published or is proprietary, basic information about inclusion and exclusion criteria for the parent study is important to provide (age and sex of participants, BMI inclusion/exclusion, if any, comorbidity or medications, for a start). It is curious why the author recorded only night time EDA. Why not perform a subjective, daytime hot flash assessment concurrent with the wearing of the monitor to assess the relationship better? The presentation would benefit greatly from a more quantitative assessment of the agreement between night time hot flashes that disrupted the participant’s sleep and objectively determined hot flashes using the monitor. The Figures do not convey information with sufficient clarity. It appears that two days of EDA data are overlaid, yet we do not know whether these were nights with vasomotor symptoms or not. It appears to be assumed that a rise in EDA means the same thing as a hot flash. How can the author be sure of this? The presentation of two years’ data in Figure 2 is also unclear. In terms of scholarship, the author does not include past literature on skin conductance monitors that have been tried (Newton KM, et al. Methods for the Design of Vasomotor Symptom Trials: The MsFLASH Network1 and the Bahr DE, et al. Miniature ambulatory skin conductance monitor and algorithm for investigating hot flash events2 are two examples the come immediately to mind). There is debate within the field as to the helpfulness of objective hot flash measurements using skin conductance. Many clinicians and patients believe that, since subjective symptoms drive treatment-seeking, objective assessments are relatively moot. The author should at least attempt to address this limitation in the manuscript.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3442", "date": "19 Feb 2018", "name": "Darrell Ricke", "role": "Author Response", "response": "The article on the parent study has now been released: Ricke DO, Harper J, Shcherbina A et al. Integrated Biomedical System [version 1; referees: awaiting peer review]. F1000Research 2018, 7:162 (doi: 10.12688/f1000research.13601.1). Details on age, sex of participants, BMI inclusion/exclusion, comorbidity, medications, etc. are not included as the parent study was not focused on vasomotor symptoms. As noted in the text \"The volunteer did not take hormone or nonhormonal formulations for the treatment of vasomotor symptoms.\" This volunteer is female as noted in the text. Accurate detection and recording of night time vasomotor symptoms while sleeping is difficult to quantify outside of laboratory polysomnographic studies. The article includes quantitative assessment of sleep impact derived from the Basis watch sleep monitoring: \"A review of all sleep time data indicates an increase in sleep interruption minutes as reported by Basis (52/1957=2.9% for EDA range 10-15 μS/cm and 15/311=4.8% for EDA range 15-25 μS/cm compared to 3823/258119=1.5% for EDA < 1 μS/cm).\" Inclusion of self-reported sleep quality like Pittsburgh Sleep Quality Index (PSQI) was not included in the original study design, but would have been a nice addition for interested volunteers. Figure 1 illustrates eight nights flanking immediately before and during the first nights that the volunteer became self-aware of vasomotor symptoms occurring. Figure 2 illustrates fluctuations in night time vasomotor symptoms spanning a two-year format in both a heat map and a summary graph with peak EDA measurements. Data are available for download for closer examinations. The article by Newton et al. evaluates and rejects three monitors as being unsuitable. The article by Bahr et al. describes another device that attaches over the sternum with adhesive hydrogels; removal of this device after a week can cause skin irritations. These articles would be good additional articles to cite. The reviewer raises the question of subjective symptoms and the drive for treatment-seeking. This article includes only quantified measurements. This volunteer did not seek treatments. In contrast the volunteer felt empowered being able to confirm symptoms with EDA measurements via Basis provided phone and web interfaces to personal measurements." } ] }, { "id": "33626", "date": "01 May 2018", "name": "JoAnn E Manson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article by D.O. Ricke provides details of the experience of an individual research participant in a longitudinal study that involved wearing physiological monitors, allowing a comparison of personal tracking of nighttime vasomotor symptoms (VMS) with the parallel capture of VMS data via the monitor’s recording of electrodermal activity. This is essentially a case report of a single individual’s experience/data and serves as a “proof of principle”. The report achieves its goal for this limited purpose; however, additional information about the background study leading to this report, the participant’s daily (daytime and nighttime) frequency and severity/”bother” of VMS if available (the report focuses on night-time tracking), and any use of medications for VMS by the patient would be helpful for the reader. Also, the literature review is limited, especially in terms of reviewing other investigators’ experience with objective monitors for VMS (results have not always been concordant with subjective reports). Readers would benefit from an expanded reference list addressing prior relevant research on this topic and from the author’s recommendations regarding integrating and/or reconciling data that may conflict from patient-reported and objective monitor sources.\n\nNonetheless, the Research Note does provide a compelling illustration of the potential utility of commercial fitness devices, which have the potential to advance our understanding of the correlates, lifestyle/behavioral “triggers” of VMS, and the response to interventions/treatments. The present study does make a contribution in showing that high resolution data captured from these devices may add to patient-reported methods such as diaries, particularly for nighttime events. Additional research is clearly needed to follow up on this N=1 study; such research will fill important gaps and will ultimately support (or refute) the value and utility of the approach.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2155
https://f1000research.com/articles/6-1898/v1	27 Oct 17	{ "type": "Research Article", "title": "Predictive value of early postoperative IOP and bleb morphology in Mitomycin-C augmented trabeculectomy", "authors": [ "Hamed Esfandiari", "Mohammad Pakravan", "Nils A. Loewen", "Mehdi Yaseri", "Hamed Esfandiari", "Mohammad Pakravan", "Mehdi Yaseri" ], "abstract": "Background: Our aim was to determine the predictive value of postoperative bleb morphological features and intraocular pressure (IOP) on the success rate of trabeculectomy. Methods: In this prospective interventional case series, we analyzed for one year 80 consecutive primary open angle glaucoma patients who underwent mitomycin-augmented trabeculectomy. Bleb morphology was scored using the Indiana bleb appearance grading scale (IBAGS). Success was defined as IOP ≤15 mmHg with or without medications at 12 months. We tested for IOP and bleb morphological differences between groups, applied a multivariable regression analysis and determined the area under the receiver operating characteristic curve (AUC). Results: Age and gender were equally distributed (62.3±13.1 years, P=0.911). IOP of patients with a successful outcome did not differ from unsuccessful ones on day 1, 7 and 30 (all P≥0.2). The AUC of IOP at day 1, day 7 and 30 for predicting a successful outcome was 0.355, 0.452, and 0.80, respectively. The AUC for bleb morphology parameters, bleb height, extension, and vascularization, on day 14 were 0.368, 0.408, and 0.549, respectively. Values for day 30 were 0.428, 0.563, and 0.654. IOP change from day 1 to day 30 was a good predictor of failure (AUC=0.838, 95% CI: 0.704 to 0.971) with a change of more than 3 mmHg predicting failure with a sensitivity of 82.5% (95% CI: 68 to 91%) and a specificity of 87.5% (95% CI: 53 to 98%). Conclusions: The postoperative IOP on day 30 had a fair to good accuracy while the bleb features failed to predict a successful outcome. An IOP increase by as little as more than 3 mmHg during the first 30 days was a good predictor of failure.", "keywords": [ "trabeculectomy", "mitomycin C", "bleb morphology", "Indiana bleb appearance grading scale", "intraocular pressure" ], "content": "Introduction\n\nFor several reasons, the number of trabeculectomies has sharply declined in the last decades in many developed countries1,2. More surgeons are now trained in microincisional glaucoma surgeries3,4 which can be performed sooner due to a superior risk profile5; laser trabeculoplasty devices have become more affordable and are used as the first line of treatment6,7; and prostaglandin analogues are now available as generic, less expensive eye medications, further reducing the number of trabeculectomies6. However, in economies categorized as developing by the International Monetary Fund8 and countries not in the upper quartile of the Human Development Index9, trabeculectomies remain the leading surgeries for moderate and severe glaucomas10,11. Although trabeculectomies have a considerable complication rate12, they remain a common primary procedure for advanced glaucoma because they lower intraocular pressure (IOP) effectively and are not as expensive as epibulbar glaucoma drainage devices13, which involve a comparable surgical effort and risk profile.\n\nCareful patient selection and postoperative management are key to making trabeculectomies successful. Recognized risk factors are, among others, younger age, a higher baseline IOP, and inflammation14,15. While meticulous preoperative planning, intraoperative use of antifibrotics, and the surgical technique impact the outcome, the postoperative period plays an equally important role16,17 including adjustment of the frequency of postoperative visits, adjustment of steroid frequency, release or lasering of scleral flap sutures, bleb massage, and bleb needling. Yet, even with the most personalized postoperative care, the individual wound healing and tissue remodeling make predicting a successful outcome difficult. Several recent studies have tried to capture the specific wound healing by correlating the early postoperative IOP or the bleb morphology with long-term outcomes, and found a low early postoperative IOP produces favorable long-term outcomes18–21. In comparison, bleb grading systems are more complex, less objective but hold promise to describe data otherwise difficult to quantify; they have not been widely adopted22,23.\n\nThe purpose of our study was to validate in our patient population the accuracy of the early postoperative IOP and bleb morphology (Indiana Bleb Appearance Grading Scale (IBAGS)) as a test to predict a failure of trabeculectomy at 12 months. In contrast to prior studies, we also examined the role of an IOP increase during the first 30 postoperative days in predicting such failure.\n\n\nMethods\n\nEighty eyes of 80 consecutive patients diagnosed with primary open angle glaucoma were included in this prospective observational study. In patients who had trabeculectomies in both eyes, the eye was chosen by randomization24. The study was performed at the glaucoma clinic of the Labbafinejad Medical Center of the Shahid Beheshti University of Medical Sciences, Tehran, Iran, from September 2013 to March 2015. It was approved by the ethics committee and the institutional review board at the Ophthalmic Research Center (protocol number: IR.SBMU.ORC.REC.1393.2) and followed the tenets of the Declaration of Helsinki. Informed written consent was obtained from each participant. Inclusion criteria were equal to or above 30 years of age and progressive primary open angle glaucoma (glaucomatous optic neuropathy, retinal nerve fiber layer loss and corresponding visual field defect on standard automated perimetry) that could not be controlled medically. Exclusion criteria consisted of prior ocular surgery, manipulation of the conjunctiva, a necessity for combining trabeculectomy with cataract extraction, ocular or systemic comorbidities that could affect the procedure and study outcomes including immunodeficiency, connective tissue disease, and uncontrolled diabetes. At baseline, all patients underwent a comprehensive ophthalmic examination including determination of best-corrected visual acuity (BCVA), slit lamp examination, Goldmann applanation tonometry, gonioscopy, and fundus examination.\n\nAll trabeculectomies were performed by two surgeons in equal numbers (HE and MP), as described in the following25. After administration of intravenous sedation, a peribulbar block was placed using 2 milliliters (ml) of 2% lidocaine (Lignidic 2%, Caspian Tamin Pharmaceutical Co., Rasht, Iran). Following lid speculum insertion and cul de sacs irrigation with povidone-iodine and normal saline solution, a 7-0 silk traction suture was placed through the superior peripheral cornea. A superior peritomy was fashioned over 1-½ clock hours, and Tenon’s capsule was dissected with Westcott scissors. A 3.0 × 4.0 mm trapezoidal half-thickness scleral flap was fashioned using a crescent knife followed by lamellar dissection of the flap 1mm into the clear cornea. After creating the scleral flap, small pieces of 0.02% MMC (Mitomycin-C, Kyowa, Kogyo Company, Tokyo, Japan) soaked cellulose sponges were placed under the Tenon and conjunctiva over the scleral flap for 3 minutes. After removing the sponges and vigorously irrigating the area with 50 ml of balanced salt solution (BSS), the anterior chamber was entered with a small keratome, and a block of clear cornea was removed with a 1 mm Kelly punch followed by a peripheral iridectomy using Vannas scissors. The scleral flap was then secured using two 10-0 nylon releasable sutures. The anterior chamber was formed by injection of BSS a paracentesis, and the filtration was titrated by adjusting the tension of the scleral flap sutures. The conjunctiva was closed using 10-0 nylon wing sutures. Cefazolin 50 mg (Cefazolin 500, Exir Pharmaceutical Co, Tehran, Iran) and betamethasone 2 mg (Betazone, Caspian Tamin Pharmaceutical CO., Rasht, Iran) were injected subconjunctivally into the inferior fornix, and the eye was patched.\n\nThe postoperative regimen consisted of chloramphenicol 0.5% eye drops (Sina Darou Lab. Co., Tehran, Iran) 4 times a day for 1 week and betamethasone 0.1% eye drops (Sina Darou Lab. Co., Tehran, Iran) 6 times a day, which was tapered over 8 to 12 weeks depending on the inflammation. The releasable sutures were removed as needed after 72 hours depending on bleb appearance to achieve IOPs near 10 mmHg. Needling was done during the postoperative period for impending failure from a contracting bleb. Thirty minutes after injecting 0.1 ml of 0.02% Mitomycin-C into the bleb-adjacent subtenon space a 27 gauge needle was used to reform the bleb and dissect adhesions at the slit lamp.\n\nWe saw our patients on a weekly basis for one month and then monthly for 12 months. At each postoperative visit, BCVA, IOP, bleb morphology, glaucoma medications, and complications were noted. A slit lamp photograph of the blebs was obtained at each visit (Haag-Streit BX 900 slit lamp, Haag-Streit Diagnostics, Köniz, Switzerland). Two glaucoma surgeons, masked to surgeon and patient, graded the bleb images on the Indiana Bleb Appearance Grading Scale (IBAGS). The IBAGS variables included the bleb height (H0-H3), extent (E0-E3), vascularity (V0-V4), and leakage (S0-S2)23. A trabeculectomy was considered successful if IOP was equal or less than 15 mmHg and more than 5 mmHg with or without glaucoma medications18.\n\nData was described as mean ± standard deviation (SD), median, range, 95% confidence interval (95% CI) and frequency (percentage). To evaluate for difference between the two groups we used the t-test, Mann-Whitney, Chi-square and Fisher exact test. A change in visual acuity before and after surgery was evaluated by the Wilcoxon signed-rank test. The predictive power of bleb variables was measured with the area under the receiver operator characteristic curve (AUC). To find the relative risk (RR, Supplementary Table 1) of failure by bleb morphology and IOP at the 1-month visit, a Cox regression with defined constant time was used. Youden's J statistic was used to capture the performance of variables (bleb morphology, IOP) as a diagnostic test. All statistical analyses were done with the SPSS software package (IBM Corp. Released 2016. IBM SPSS Statistic for Windows, Version 24.0, Armonk, NY).\n\n\nResults\n\nA total of 80 patients were enrolled in this study. This sample size and ratio of success and failure provided an above 90% power to detect an AUC difference of 0.526. Forty-four eyes would have provided a power of 80% to detect an IOP change of 3 mmHg while 80 eyes allowed for a 97% power. The mean age of study participants was 62.3±13.1 years, and 51.3% of the patients were male (P=0.911). The characteristics of participating patients are presented in Table 1. The procedure was considered successful in 64 patients (80%) at 12 months follow-up.\n\nBCVA: Best Corrected Visual Acuity. Medications: topical glaucoma medications. IOP: Intraocular Pressure. HVF MD: Humphrey visual field mean deviation. VCD: vertical cup disc ratio.\n\n† Based on t-test.\n\n‡ Based on Mann-Whitney test.\n\nBased on Chi-Square test.\n\nPreoperative medications in the no success group were 1.4±1.9 and in the success group were 1.2±1.6 (p=0.785), while the postoperative medications were 1.4 ± 0.5 in the no success group and 1.0±0.0 in the success group (p=0.04). There were no significant differences between the success and failure groups in terms of age, the number of preoperative glaucoma medications, preoperative IOP, visual field mean deviation or vertical cup-disc ratios (Table 1). The postoperative IOP was not different between these groups at any time (Table 2) because an IOP of lower than 5 mmHg was also counted as no success in accordance to prior bleb rating studies. When such a low IOP was considered a success in the absence of hypotony maculopathy, the average IOPs was higher at month 12 (18.9 ± 2.3 [no success] and 9.8 ± 2.1 [success], p<0.001, Supplementary Table 1).\n\nThe mean and standard deviation, median and range and area under the receiver operating characteristic curve (AUC) with lower and upper confidence interval (CI) are shown for all timepoints. A higher AUC indicates a better ability to distinguish between the two groups.\n\n‡ Based on Mann-Whitney test.\n\nThe best corrected visual acuity did not change significantly (0.47±0.47 logMAR at baseline, 0.48±0.48 postoperatively, P=0.492). Mean IOP decreased significantly from 21.8±5 mmHg at baseline to 10.6±3.8 mmHg at 12 months (p<0.001), and 25 patients needed glaucoma medications to have their IOP within the target range. There were no intraoperative complications. The most common postoperative complication was a shallow anterior chamber, followed by bleb leakage and hyphema as summarized in Table 3.\n\n* Based on Fisher exact test.‡ Based on Mann-Whitney test.\n\nThe difference between success and failure groups in a leak, repeat suture, hyphema and shallow anterior chamber were insignificant (P=0.09, 0.99, 0.59, and 0.18, respectively). Bleb needling was more frequently performed in the group that failed (P<0.001). One eye experienced hypotony maculopathy. Patients with a ﬁnal IOP of ≤15 mmHg had a mean day 1 IOP of 7.4±2.5 mmHg compared with 6±2.4 mmHg in those with an IOP above 15 mmHg at ﬁnal follow-up (P=0.193). Respective values for day 7 were 7.8±2.5 and 7.1±2.1 mmHg (P=0.639), and for day 30 values were 8.4±2.4 and 9.3±4.6 mmHg (P=0.238). The AUC for IOP at day 1, day 7 and 30 as a predictor of success was 0.355, 0.452, and 0.80 respectively. The bleb morphology parameters according to the IBAGS are detailed in Table 4.\n\nH: height. E: extension. V: vascularization.\n\n‡ Based on Mann-Whitney test.\n\nThe AUC for bleb morphology parameters of bleb height, extension, and vascularization on day 14 were 0.368, 0.408 and 0.549, respectively. The AUC values for day 30 were 0.428, 0.563, and 0.654. Based on the AUCs, there was no single bleb variable at month 1 to predict success at month 12.\n\nSurprisingly, the AUC of the IOP change from day 1 to day 30 was a good predictor of success (AUC=0.838, 95% CI: 0.704 to 0.971, Supplementary Table 1). Based on Youden’s J index, the change in IOP less than or equal to 3 mmHg could predict the success in month 12 with the sensitivity of 82.5% (95% CI: 68 to 91%) and specificity of 87.5% (95% CI: 53 to 98%).\n\n\nDiscussion\n\nTrabeculectomy remains a common surgery to lower IOP in glaucoma patients and has a long history. In 1958, Grant was the first one to describe a guarded filtering procedure in enucleated eyes with subsequent facility measurements which he termed trabeculectomy27. Sugar followed by performing the first such trabeculectomy in patients in 196128 and Cairns finally popularized the term and technique by providing detailed drawings and figures29. The procedure was eventually made more effective by adding Mitomycin-C as an antifibrotic30 and somewhat titratable by using sutures that can be lasered or manually released31,32. The variable success rate of this procedure can be improved by careful patient selection, surgical technique33, and postoperative management15. Identifying patients at risk is critical because a timely intervention can improve the outcomes of trabeculectomy16; preoperative factors including high baseline IOP, number of glaucoma medications, young age, aphakia or pseudophakia and prior surgery with conjunctival scarring are known risk factors for failure33. The success and failure groups in our study have similar baseline IOP, medications, visual field loss and optic nerve damage indicating that any risk factors related to those were relatively equal in both.\n\nSeveral recent studies found a low early postoperative IOP produces favorable long-term outcomes18–21,34 but also causes more frequent and more severe complications that include a shallow anterior chamber, choroidal effusion, hypotony maculopathy, and vision loss35. Although we did observe that the IOP change during the first month was a good predictor of failure, we found only a weak relationship between absolute IOP at day 1, 7, and 30 and long term success. This is similar to Polikoff et al.36 who also reported that early post-trabeculectomy IOP is a poor predictor of success at one year follow-up. The accuracy of a test depends on how well it separates groups under study into those with and without the issue in question. Accuracy is measured by the area under the ROC curve and is commonly classified into .90-1 = excellent, .80-.90 = good, .70-.80 = fair, .60-.70 = poor and .50-.60 = fail26. In our study, the AUC for IOP was the highest on day 30 with 0.8, making it a fair to good predictor26. Early post-trabeculectomy IOP is affected by various factors that include reduced aqueous production, inflammation, the amount of TGF-beta and other growth factors37, and breakdown of the blood-aqueous barrier, over filtration, and choroidal detachment. In our study, the IOP appears to summarize these and other factors reasonably well to a single variable that can be used as a predictor.\n\nAn advantage of this study is that approximately 75% more patients participated than in prior bleb grading studies22,23,38,39. Different from those, we also examined the change in IOP during the first 30 days and found that it served as a good predictor of success that performs better than the IOP itself. The cutoff for an at-risk IOP elevation was only 3 mmHg. Our study was not designed to discover causality, however. It is likely that this finding identifies patients at risk of failure who experienced an IOP increase despite an intensified treatment. It could also indicate that these were patients who received a suboptimal postoperative management. An elevated IOP will increase the stretch of the bleb wall and incite a reactive fibrosis. Stretch is a well-established activator of fibroblast proliferation and fibrosis40,41. This may lead to a cycle of auto-enhancing IOP increase with further increase of stretch and fibrosis that again elevates pressure as the bleb wall becomes thicker and the bleb contracts. In this model, IOP drives the morphological changes of the bleb. This order matches our AUC findings better (the AUC was better for IOP change than for bleb morphology) than a primary morphological change that is followed by increased IOP. Our observation is supportive of the practice to inject or needle with postoperative 5-fluorouracil or Mitomycin-C to reduce the fibrosis42,43.\n\nLike IOP, a bleb’s morphology is a reflection of a patient’s specific wound healing that is directly observable but more difficult to quantify. The development of bleb grading scales was an attempt to approach this problem systematically22,23. While grading systems can be useful teaching tools of bleb features, its inherently subjective nature and categorical data weaken the predictive power. Accordingly, we found only low AUC values for bleb morphology that ranged from 0.368 to 0.549 on day 14 and would be interpreted as a test failure. The day 30 features would be interpreted only slightly better, as poor. Of the variables bleb height, extension and vascularization, vascularization had the highest accuracy. These findings match Picht et al.44 who concluded an early vascularization indicates a poor prognosis with higher IOP at 12 months and may support use of anti-VEGF agents45,46. The above reasons and recent reports of ab interno trabeculectomy results with a reduced complication rate, considerable range of glaucoma indications, and results that can rival traditional glaucoma surgery47–55 suggest a conventional trabeculectomy with Mitomycin-C may be more appropriately used as a secondary procedure when microincisional surgical modalities are available or only be used in advanced glaucoma with a rather low IOP target.\n\nIn conclusion, we found that the IOP change during the first month, rather than the IOP at each visit, was a good predictor of failure. The bleb morphological features did not predict failure except for bleb vascularity which performed poorly to fairly. These finding highlight how important it is to carefully observe for IOP and bleb vascularity changes and to intervene swiftly if necessary.\n\n\nData availability\n\nDataset 1: Raw data collected from all study participants. DOI, 10.5256/f1000research.12904.d18128856.", "appendix": "Competing interests\n\n\n\nHE, MP, and MY have no financial disclosure.\n\nNAL has received honoraria for trabectome wet labs and lectures from Neomedix Corp.\n\n\nGrant information\n\nThis study was funded in part by the Initiative to Cure Glaucoma, of the Eye and Ear Foundation of Pittsburgh, and by the Ophthalmic Research Center of the Shahid Beheshti University of Medical Science.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Table 1: Intraocular pressure at each pre- and postoperative visit when success included IOPs below 5 mmHg without complications.\n\nClick here to access the data.\n\n\nReferences\n\nRamulu PY, Corcoran KJ, Corcoran SL, et al.: Utilization of various glaucoma surgeries and procedures in Medicare beneficiaries from 1995 to 2004. Ophthalmology. 2007; 114(12): 2265–2270.e1. 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Clin Exp Ophthalmol. 2016; 44(9): 783–788. PubMed Abstract \| Publisher Full Text\n\nDang Y, Roy P, Bussel II, et al.: Combined analysis of trabectome and phaco-trabectome outcomes by glaucoma severity [version 2; referees: 3 approved]. F1000Res. 2016; 5: 762. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang C, Dang Y, Waxman S, et al.: Angle stability and outflow in excisional ab interno trabeculectomy with active versus passive chamber management. Peer J Preprints. 2017; 5: e2762v2. Publisher Full Text\n\nEsfandiari H, Pakravan M, Loewen N, et al.: Dataset 1 in: Predictive value of early postoperative IOP and bleb morphology in Mitomycin-C augmented trabeculectomy. F1000Research. 2017. Data Source" }	[ { "id": "27402", "date": "08 Nov 2017", "name": "Ding Lin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a carefully done study containing interesting results which merit indexing. It is very useful and can provide a proof for ophthalmologist to decide how long the patient should be re-visit postoperation according to IOP change. For the benefit of the reader, however, a number of points need clarifying and certain statements require further justification. A few minor revisions are list below.\nThere are mistakes in the study results and we listed some examples in the following and you should revised it in your paper carefully and discreetly.\n1.1 In the article, they describe that collect IOP outcomes on Day 7, but it was Week 2 in table 2. In the results part of Line 3 Page 1 (All P≥0.2), but it was 0.193 in day 1 in table 2.\n1.2 IOP change more than 3mmHg in Page 1, but it was less than or equal than 3 mmHg in Page 6.\n\nThere are some misunderstanding in the article.\n2.1 The first sentence in the results of page 1. (Age and gender were equally distributed (62.3±13.1 years, P=0.911).)\n2.2 The anterior chamber was formed by injection of BSS a paracentesis, and the filtration was titrated by adjusting the tension of the scleral flap sutures.(Page 3).\n\nThe releasable sutures were removed as needed after 72 hours depending on bleb appearance to achieve IOPs near 10 mmHg. (Page 3, Marked in yellow and underline). In what time the releasable suture were removed for most of them (for example 7-10 days or something else for 70%(or ?%) patient), and you removed the two releasable suture for one time or two time respectively. If you removed the suture for two times, how much time will pass between them.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3221", "date": "19 Dec 2017", "name": "Nils Loewen", "role": "Author Response", "response": "1. There are mistakes in the study results and we listed some examples in the following and you should revised it in your paper carefully and discreetly. 1.1 In the article, they describe that collect IOP outcomes on Day 7, but it was Week 2 in table 2. In the results part of Line 3 Page 1 (All P≥0.2), but it was 0.193 in day 1 in table 2. Authors: We very much appreciate taking the time and effort to assess our manuscript. We are grateful for the many good suggestions. There was indeed a discrepancy in the Results section. It should be week 1 in the table as we now write correctly. We have confirmed this in our source SPSS database file. Regarding the P values in the abstract, we replaced >0.2 in the sentence: “IOP of patients with a successful outcome did not differ from unsuccessful ones on day 1, 7 and 30 (all P≥0.2).“ with specific P values for each time point: “IOP of patients with a successful outcome did not differ from unsuccessful ones on day 1, 7 and 30 (P= 0.193, 0.639, and 0.238, respectively.)” 1.2 IOP change more than 3mmHg in Page 1, but it was less than or equal than 3 mmHg in Page 6. Authors: That is correct but there is no real contradiction. The reason for this is that we talk about that a change in IOP less than or equal to 3 can predict the success at month 12. In contrast, we talk about failure when a change of more than 3 mmHg was observed. 2. There are some misunderstanding in the article. 2.1 The first sentence in the results of page 1. (Age and gender were equally distributed (62.3±13.1 years, P=0.911).) Authors: Thank you for pointing this out. We changed the sentence \"Age and gender were equally distributed\" to \"The mean age of the study participants was 62±12.3 years in the success and 63.2±16.3 years in the failure group (P= 0.430) with equal gender distribution (P=0.911)\". 2.2 The anterior chamber was formed by injection of BSS a paracentesis, and the filtration was titrated by adjusting the tension of the scleral flap sutures. Authors: We apologize and have changed the sentence to state “The anterior chamber was formed by injection of BSS a paracentesis, and the filtration was titrated by adjusting the tension of the scleral flap sutures “to “The anterior chamber was formed by injection of BSS through a sideport, and the filtration was titrated by adjusting the tension of the scleral flap sutures\". 3. The releasable sutures were removed as needed after 72 hours depending on bleb appearance to achieve IOPs near 10 mmHg. (Page 3, Marked in yellow and underline). In what time the releasable suture were removed for most of them (for example 7-10 days or something else for 70%(or ?%) patient), and you removed the two releasable suture for one time or two time respectively. If you removed the suture for two times, how much time will pass between them. Authors: We changed Methods to state: \"In most cases, the releasable sutures were removed within 4 weeks after the surgery. One removed first and if the IOP was not within the target range the other one was released during at the follow up one week later. Needling was done during the postoperative period for impending failure from highly vascularized and contracting blebs\"." } ] }, { "id": "27865", "date": "28 Nov 2017", "name": "Frances Meier-Gibbons", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe study is carefully done and the topic is interesting for clinicians.\n\nComments/remarks:\n\nYou write the exact number of preoperative and postoperative medications the patients used. As there are differences in the IOP-lowering effects of the different types of these drugs, it would be interesting to see what type of IOP-lowering drugs the patients applied and if there was a difference in the type of drugs used pre- and postoperatively.\n\nYou state that you used postoperative needling with Mitomycine C, but you do not clarify when exactly (\"impending failure from a contracting bleb\" is not specific). Please give a comment in which patients you used the needling and add also in how many patients a needling was necessary.\n\nIn the \"Conclusions\" of the abstract you state that \"the bleb features failed to predict a successful outcome\" while in the \"Conclusions\" at the end of the manuscript you say that an augmented vascularity in the bleb should be carefully observed. Please add this sentence in the \"Conclusions\" of the abstract.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3222", "date": "19 Dec 2017", "name": "Nils Loewen", "role": "Author Response", "response": "The study is carefully done and the topic is interesting for clinicians.Comments/remarks:1. You write the exact number of preoperative and postoperative medications the patients used. As there are differences in the IOP-lowering effects of the different types of these drugs, it would be interesting to see what type of IOP-lowering drugs the patients applied and if there was a difference in the type of drugs used pre- and postoperatively. Authors: We very much appreciate the reviewer’s time and care reviewing our manuscript. Thank you for allowing us to clarify this point. We now state in Methods: \"Before the surgery patients used glaucoma medications from all four major classes, including beta-blockers, prostaglandin analogous, alpha-2 agonists, and carbonic anhydrase inhibitors. Postoperatively, patients received the same drops that had proved effective and safe before surgery if the target IOP was not achieved. The only exception was prostaglandins analogous that we tried to avoid during the first three postoperative months.\"2. You state that you used postoperative needling with Mitomycin C, but you do not clarify when exactly (\"impending failure from a contracting bleb\" is not specific). Please give a comment in which patients you used the needling and add also in how many patients a needling was necessary. Authors: Thank you for allowing us to clarify this point. We needled only highly vascularized and contracting blebs when the IOP was uncontrolled. We now state: \"A total of ten eyes (12.5%) underwent needling, including two eyes in the success group and eight eyes in the failure group (P=0.001).\"3. In the \"Conclusions\" of the abstract you state that \"the bleb features failed to predict a successful outcome\" while in the \"Conclusions\" at the end of the manuscript you say that an augmented vascularity in the bleb should be carefully observed. Please add this sentence in the \"Conclusions\" of the abstract. Authors: This is a good point. We have modified the Conclusions in the Abstract based on the reviewer’s comments:“Conclusions: The postoperative IOP on day 30 had a fair to good accuracy while the bleb features failed to predict a successful outcome except for bleb vascularity that predicted success with poor to fair accuracy. An IOP increase by as little as more than 3 mmHg during the first 30 days was a good predictor of failure. It is important to carefully observe for IOP changes and bleb vascularity to intervene if necessary.”" } ] } ]	1	https://f1000research.com/articles/6-1898
https://f1000research.com/articles/6-1302/v1	02 Aug 17	{ "type": "Research Article", "title": "New chimeric RNAs in acute myeloid leukemia", "authors": [ "Florence Rufflé", "Jerome Audoux", "Anthony Boureux", "Sacha Beaumeunier", "Jean-Baptiste Gaillard", "Elias Bou Samra", "Andre Megarbane", "Bruno Cassinat", "Christine Chomienne", "Ronnie Alves", "Sebastien Riquier", "Nicolas Gilbert", "Jean-Marc Lemaitre", "Delphine Bacq-Daian", "Anne Laure Bougé", "Nicolas Philippe", "Therese Commes", "Florence Rufflé", "Jerome Audoux", "Anthony Boureux", "Sacha Beaumeunier", "Jean-Baptiste Gaillard", "Elias Bou Samra", "Andre Megarbane", "Bruno Cassinat", "Christine Chomienne", "Ronnie Alves", "Sebastien Riquier", "Nicolas Gilbert", "Jean-Marc Lemaitre", "Delphine Bacq-Daian", "Anne Laure Bougé", "Nicolas Philippe" ], "abstract": "Background: High-throughput next generation sequencing (NGS) technologies enable the detection of biomarkers used for tumor classification, disease monitoring and cancer therapy. Whole-transcriptome analysis using RNA-seq is important, not only as a means of understanding the mechanisms responsible for complex diseases but also to efficiently identify novel genes/exons, splice isoforms, RNA editing, allele-specific mutations, differential gene expression and fusion-transcripts or chimeric RNA (chRNA). Methods: We used Crac, a tool that uses genomic locations and local coverage to classify biological events and directly infer splice and chimeric junctions within a single read. Crac’s algorithm extracts transcriptional chimeric events irrespective of annotation with a high sensitivity, and CracTools was used to aggregate, annotate and filter the chRNA reads. The selected chRNA candidates were validated by real time PCR and sequencing. In order to check the tumor specific expression of chRNA, we analyzed a publicly available dataset using a new tag search approach. Results: We present data related to acute myeloid leukemia (AML) RNA-seq analysis. We highlight novel biological cases of chRNA, in addition to previously well characterized leukemia chRNA. We have identified and validated 17 chRNAs among 3 AML patients: 10 from an AML patient with a translocation between chromosomes 15 and 17 (AML-t(15;17), 4 from patient with normal karyotype (AML-NK) 3 from a patient with chromosomal 16 inversion (AML-inv16). The new fusion transcripts can be classified into four groups according to the exon organization. Conclusions: All groups suggest complex but distinct synthesis mechanisms involving either collinear exons of different genes, non-collinear exons, or exons of different chromosomes. Finally, we check tumor-specific expression in a larger RNA-seq AML cohort and identify new AML biomarkers that could improve diagnosis and prognosis of AML.", "keywords": [ "chimeric RNA", "RNAseq", "acute myeloid leukemia", "biomarkers", "bioinformatics analysis", "Complex Read Analysis", "Classification (CRAC)", "PML-RARA", "TRIM28" ], "content": "Introduction\n\nHigh-throughput sequencing technologies (NGS) enable the detection of new biomarkers used for tumor classification and disease monitoring, including patient response to therapies. Whole-transcriptome analysis with RNA-seq is increasingly acquiring a key role, not only to learn about mechanisms responsible for complex disease, but also to identify novel genes/exons, splice isoforms, RNA editing, allele-specific mutation, differential gene expression, fusion-transcripts and chimeric RNA (chRNA)1,2.\n\nFor chimeric RNA, a group of fusion transcripts is increasingly used by geneticists in oncology diagnosis3,4. These cancer biomarkers are generated at DNA level from gene fusions by mechanisms such as translocations, inversions, or more complex chromosomal rearrangements. In some well-documented cases, gene fusions, in addition to contributing to neoplastic transformation, produce fusion RNA and proteins used as therapeutic targets (5,6 and references therein). However recent RNA-seq analyses have revealed the existence of an enlarged “chimeric transcriptome”5–10 generated by new RNA processing events such as cis- and trans-splicing, whose mechanisms and functional roles are poorly understood. It is crucial to determine whether these events represent artefacts of RNA-sequencing, transcriptional noise with little impact on cell functions or tissue-specific transcripts, or whether these events are important in tumor development. Several significant examples of chRNA without corresponding fusions at DNA level have been described in the context of neoplasia11,12.\n\nBoth from a methodological and biological perspective, profiling chimeric RNA is a challenging issue. chRNA’s distinctive features must be identified to provide relevant biological information, including synthesis mechanisms and pertinence as a biomarker. Though RNA-Seq may enable new biomarker discovery, there is a lack of consensus on which analysis tool and algorithmic strategy should be used, especially in the detection of chRNA13. We recently proposed Crac14, a novel way of analyzing reads that combines genomic locations and local coverage to classify the biological events, to directly infer splice and chimeric junctions within a single read. The Crac software is based on an innovative algorithm that allows extraction of transcriptional events, irrespective of annotation. The main advantage of using Crac to detect chimeras is its high sensitivity and specificity, allowing detection of rare events with confidence. We developed the complementary CracTools module to aggregate, annotate, and filter the chRNA reads. The procedure classifies reads into 4 classes, depending on the exon organization. The first class corresponds to fusion transcripts arising from two different chromosomes, the second class includes parts of two genes belonging to the same chromosome strand. The third and fourth classes involve non-collinear transcription on the same chromosome. This categorization suggests that each class represents distinct, complex synthesis mechanisms.\n\nIn order to assess Crac’s potential to identify new biomarkers, we present data related to acute myeloid leukemia (AML) RNA-seq analysis. AML provides a practical cellular model to detect chRNA biomarkers in order to improve classification and patient follow-up in precision medicine2. Despite the fact that leukemia chRNAs are well characterized, our study highlights new biological cases of chRNA. We identify and validate 17 chRNAs initially detected in 3 AML patients that belong to the 4 classes. We then explore their specific expression in the publicly available LEUCEGENE cohort15, and propose new criteria for distinguishing chRNAs based on their recurrence, tumor, subgroup, or patient-specific expression. We also classify the chRNAs according to differences in expression of the genes linked to the chimera, in healthy donors vs AML patients. Finally, we identify new biomarkers that could improve diagnosis and prognosis of AML.\n\n\nMaterials and methods\n\nThree sets of samples from patients with AML were used in this study. Each patient is designed by a specific ID including the OM or OS code corresponding respectively to bone marrow or blood sample (Supplementary Table S1). The first set of 25 AML samples consisted of 11 AML-NK samples, 4 AML-inv16, 5 AML-UK, 2 AML-t(15; 17) also called Acute Promyelocytic Leukemia (APL), and 3 other AML-AK samples (Table S1 lines 3 to 27). They were supplied by JBG (Biological Resource Center CHU-Nîmes, France) and included both RNA and peripheral blood mononuclear cells (PBMC), stored in RNALater (Ambion, USA) according to the manufacturer instructions. The second set of 14 AML samples consisted of 10 AML-UK, 2 AML-NK, and 2 AML AK. They were supplied by AM (Medical Genetic Unit, University St Joseph, Lebanon) (Table S1 lines 28 to 41). They included blood and bone marrow stored in TRIzol reagent (Life Technologies, USA). The third set of AML samples included 3 AML-t(15;17), and were provided by CC and BC (Cell Biology Unit, Hôpital St-Louis, France). For the latter, peripheral blood mononuclear cells were collected by ficoll-hypaque density gradient and cultured at a concentration of 1×106/ml, with or without 0,1µM ATRA, for 3 days. PBMC samples from healthy donors were pooled and used as control sample. The patients and healthy donors provided written informed consent to participate in the study, in accordance with the Declaration of Helsinki. The U937 leukemia cell line (DSMZ, Braunschweig, Germany), NB4 promyelocytic cell line and NB4-LR2 cell line (provided by CC) were cultured in RPMI 1640 (Invitrogen, USA) containing 10% decomplemented FBS (Dutscher, Brumath, France). For differentiation conditions, NB4 and U937 cells were cultured as previously described16,17. The chemical agents used for differentiation were 1µM all-trans retinoic acid (ATRA; Sigma-Aldrich, Gillingham, UK), or 0,1µM vitamin D (VD) associated with 500pg/ml transforming growth factor beta TGFβ (Promega Corporation, USA) for the NB4 cell line18. For the U937 cell line, 0,1µM TTNPB associated with 1µM Targretin (LGD1069) and 0,1µM 1 alpha, 25 dihydroxyvitamin D3 (VD) were used. TTNPB, LGD1069 and VD were kindly provided by Dr Klaus (Hoffman-La Roche,Switzerland), JHiernaux (Glaxo-welcome Laboratories, France), and L Binderup (Leopharmaceutical products,Denmark), respectively. We also used human neuroblastoma cancer SH-SY5Y cells, human breast cancer MCF7 cells and human prostate cancer MDA-PCa cells. Cell pellets were kindly provided by S. Marchal (University Montpellier, France), and by D.Noel (Institute of Regenerative Medicine and Biotherapies, France) for the latter.\n\nRNA was extracted with the RNeasy Qiagen kit (Qiagen, Germany), additional DNase treatment was performed in order to remove residual DNA (RNAse free DNase set, Qiagen, Germany). Total RNA was quantified using a NanoDrop® ND-1000 spectrophotometer (NanoDrop ND-Thermo Fisher Scientific, USA). RNA quality and quantity were assessed using the 2100-Bioanalyzer (Agilent Technologies, Waldronn, Germany). Reverse transcription was performed with random primers and MultiScribe Reverse Transcriptase (High-capacity cDNA Archive kit; Applied Biosystems, USA), using 1 µg of total RNA. To check for possible chRNA formation induced by transcriptional artefacts, part of the samples were double reverse transcribed. In this case, the second reverse transcription reaction was performed with ImProm-II™ Reverse Transcriptase (Improm-II Reverse Transcription System, Promega, USA).\n\nThree AML samples (AML-NK, AML-t(15;17) and AML-inv16) taken from patients OM100011, OM110223 and OS110089, respectively (AML test group) from the Biological Resource Center, CHU-Nîmes, France, were selected for RNA-seq experiments. 4 µg of total RNA taken from bone marrow (OM100011, OM110223) or blood PBMCs (OS110089) were sent to GATC biotech and analyzed on an Illumina HiSeq 2000 to generate 100 base pairs of stranded RNA-Seq paired-end reads. The following publicly available datasets of the LEUCEGENE projectdedicated to Acute myeloid and lymphoid leukemia studies15, have been used in this study (a detailed list is provided in Supplementary Table S6):\n\nGSE48846 (17 CD34 hematopoietic stem cell),\n\nGSE49642 part1 (43 AML-NK),\n\nGSE62190 part3 (82AML-AK with 24 inv16, 19 t(8;21),5 inv3,7 t(9;11), 5 t(6;11),6 t(11;19) and complex karyotypes); and\n\nENCODE publicly available datasets (Supplementary Table S7).\n\nRNA-seq analyses were performed serially using Crac (V2) and CracTools (V1.2)14. Crac is a software for analyzing reads when a reference genome is available, that is completely independent of annotations. It ignores the sequence quality of reads and classifies reads by detecting diverse biological events (mutations, splice junctions, and chRNAs) and sequencing errors from a RNA-seq read collection. In this analysis, we used two Crac versions in succession (V1.6 and V2) to extract and classify chRNAs, with the GRCh37/hg19 genome as reference genome. Crac extracts the chimeric reads supporting the chimeric junction (spanning junction) made of a non-collinear arrangement of genomic regions14. CracTools was then used to aggregate, annotate and filter the chRNA reads and extract the chimeric paired reads (spanning PE) (Figure S1). Reads were annotated according to a GFF file from ENSEMBL Genome Browser (link to GFF file in Data Availability section) by giving priority to location in exons of the annotated genes. The GFF file was built from ENSEMBL (Ensembl 84 annotations). When reads were located on a non-annotated, transcribed region, the corresponding “NONE” annotation was mentioned. The procedure included classifying chRNA into four categories depending on exon organization, as described in the introduction. This classification resembles the one depicted in 7 and can be summarized as follows:\n\n– Class 1, the exons are located on different chromosomes;\n\n– Class 2, the exons are collinear but most likely belong to different genes, to be verified through the annotation;\n\n– Class 3, the exons are on the same chromosome and same strand, but not in the order in which they are found on DNA;\n\n– Class 4, the exons are on the same chromosome but on different strands.\n\nFor each analyzed chimera, the pipeline provides related information, including a unique read identifier annotated by the pipeline, class, number of spanning junctions and spanning reads:\n\n1. ID: A unique read ID for each chimera, composed of ‘sample name: chimera ID’\n\n2. Fusion gene names (left-right)\n\n3. Chr(left): Chromosome number of the 5' part of the chimera\n\n4. Pos1: Genomic position of the 5' part of the chimera\n\n5. Strand1: Genomic strand of the 5' part of the chimera (+1 or -1).\n\n6. Chr(right): Chr number of the 3' part of the chimera\n\n7. Pos2: Genomic position of the 3' part of the chimera\n\n8. Strand2: Genomic strand of the 3' part of the chimera\n\n9. ChimValue: the chim-value takes into account methodological parameters and ambiguities, including the read mapping (P_loc) and the read coverage (P_support).13\n\n10. Spanning junction normalized: Spanning junction reads coverage (normalized per billion of reads). A spanning junction read is the read that contains the chimeric junction.\n\n11. Spanning PE normalized: Coverage of paired-end reads (normalized per billion of reads) that contains the chimeric junction in the non-sequenced part\n\n12. Class: Chimeric class from 1 to 4.\n\nThe filtering process with CracTools considered the following thresholds:\n\na. Only candidate fusions (chRNA) with at least one read covering the fusion breakpoint,\n\nb. Spanning reads must be associated with pair-end reads,\n\nc. An annotation of the fusion junction matching almost a known expressed sequence (gene A or Gene B),\n\nd. A ChimValue of up to 60, allowing the removal of false positives corresponding to pseudogenes and splicing events detected by GSNAP.\n\nCandidate fusion transcripts involving adjacent genes within a 3Kb distance region were discarded. To estimate the number of supporting reads for a chimeric candidate, CracTools extracted the count number of spanning junctions and spanning PE reads. All candidate fusion transcripts were validated using qPCR and Sanger sequencing except the class three overlap which requires systematic reconstruction of the fusion transcript for the design of primers.\n\nThe potential chimeras are listed in Table S2 with the appropriate features. For each fusion transcript, the Crac software provided a reconstructed sequence comprising, on one hand, the chimeric junction sequence based on the most representative read, and on the other hand, the paired read sequence. The symbol (#) marks the segment of the read that was not sequenced and the () symbol marks the junction point (Table S2 and Supplementary Figure S1). The sequences of reads, both junction and paired, which supported the chimera were mapped (BLAT, UCSC) to the human genome GRCh37/hg19 in order to identify complex biological events (splicing, SNPs, insertions, deletions, repeats, polymorphisms, etc.). Complementary annotations were identified usingthe ENSEMBL genome browser to determine exons and spliced variants involved in the transcript. Protein sequences and functional annotations were also verified to identify affected protein domains and to evaluate potential protein damage in selected chRNAs.\n\nReverse transcription was performed as described above. 1 µl of each cDNA sample (2ng/µl) was added to a 5 µl of reaction mix containing 3 µl of Master Mix (LightCycler®480 sybr green I Master, Roche Diagnostics, GERMANY) and 0.33 µM forward and reverse primers. Mix and cDNA were loaded onto the 384-well PCR plate using an epMotion 5070 automated pipetting system (Eppendorf, Germany). Primer sequences were designed using the Primer3Plus web interface, with some constraints as described in the PCR strategy (see Supplementary Figure S1), and synthesized by Eurofins MWG Operon, Germany. The amplification area was centered on the junction, and primers were designed to tag each sides of the junction. Primers are listed in Supplementary Table S3. PCRs were carried out in 384-well plates on a LightCycler®480 Real-Time PCR System (Roche Diagnostics, Germany). Amplifications were performed according to the following conditions: 95°C for 5 min, then 55 cycles as follows: 95°C for 10s, followed by T°C depending on Tm for 10s, and 72°C for 10s. Ultimately, a melting curve analysis ranging from 95°C to 60°C was performed to control primer specificity. For each sample, the graph of the negative first derivative of the melting curve gave a specific peak corresponding to the amplified transcript. Samples with TM value peaks different from those found in the negative control were considered potential positive targets and retained for sequencing. PCR products were purified with the Minelute PCR purification kit (Qiagen, Germany) and sequenced on the ABI 3730XL (Eurofins MWG Operon, Germany).\n\nFor the newly discovered Class1 chRNA, identified in patient OS110089 and corresponding to Chr2 and Chr13 positions and to PAN3-NONE annotations, the presence of chRNA was checked in leukemia samples obtained during the patient follow-up (Figure S3). The NONE transcript expression was checked by qPCR in human embryonic stem cells HD129 (cDNA was kindly provided by J. De Vos, Institute of Regenerative Medicine and Biotherapies, France), in AML samples and in U937, NB4, SH-SY5Y, MDA-PCa and MCF7 cell lines. To this end, we designed forward and reverse primers on the 5’NONE sequence (Figure S2).\n\nMolecular cytogenetics were performed on metaphases from bone marrow aspirate collected from the samples using a synchronised protocol. A first step aging slide with the cytogenetic preparation was performed by immerging slides in 2xSSC solution (saline sodium citrate) for 30 minutes at 37°C, followed by dehydration in 3 baths of increasing ethanol concentration: 70°, 85° and 100°, each for 1 minute. Finally, slides were air dried at room temperature. To confirm the putative t(2;13) translocation related to the PAN3-NONE fusion gene, a fusion probe was designed using bacterial artificial chromosome (BAC) technology, framing the following regions of interest: RP11-239J16, RP11-339H12 in chromosome 2p21 (labeled in Cy3 Orange) and RP11-179F17, RP11-95G6 in chromosome 13q12 (labeled in Alexa 488 Green) (BlueGnome, Cambridge, UK). 1 μl of each of the BACs was mixed and diluted in 9 μl of hybridization BlueFISH Buffer and then applied to the slide. Chromosomes fixed on the slide and probe of interest were denatured in a single step using Thermobrite (Abbot Molecular, USA). Codenaturation was done at 75°C for 5 minutes, followed by hybridization at 37°C in a humid atmosphere overnight. To remove the probe that would not properly hybridize, two successive washes in stringent conditions were performed: the first one in 0.4xSSC and 0.1% Igepal at 73°C for 2 minutes, the second one in 2xSSC and 0.3% Igepal for 1 minute at room temperature. After complete drying, 10 µl of counterstaining reagent (DAPI 125ng/ml, Abbott Molecular, Chicago, USA) was added. Slides were observed on an epifluorescence microscope (Olympus BX60). For both probes, positivity was defined as the presence of two fusion signals (co-location) in addition to a red signal and a green signal.\n\nThe tag search approach consisted of extracting representative sequences (Tags) 30 nucleotides in length, centered on the chimeric junctions. The latter were then searched in LEUCEGENE and ENCODE publicly available RNA-seq datasets. A FASTA file, listing these tags of interest, designed for each chRNA, was submitted to a specific pipeline (countTags, https://github.com/jaudoux/countTags) that searched for sequences and their reverse complement with an exact match in the FASTQ files. For each FASTQ file and each tag, the total number of tags (total k-mers) is counted. Final value is given in a delimited table, with a tag count normalized per 5 billion of k-mers.\n\nFor the gene expression clustering, chRNA were selected as below. Chimeric genes were extracted using cracTools predictions for samples OS110089, OM100011 and OM110223. Among all chimeric junctions detected, only those having a \"chimValue\" greater than 75 and with at least 3 spanning reads were conserved. Read-through chimeras were further selected based on three criteria:\n\ni. Chimera annotated as \"Class 2\" (collinear transcription),\n\nii. Short fusion distance (max 300k),\n\niii. Short exon-end distance (max 20bp).\n\nTandem repeat chimeras were further selected based on three criteria:\n\ni. Chimera annotated as \"Class 3\"\n\nii. Overlaping chimeric fragments (the two parts of the chimeric read correspond to overlapping sequences on the genome)\n\niii. Both chimeric fragments are located on the same exon of the same gene.\n\nFor each chimera type (read-through and tandem-repeat), only genes involved in at least 2 different chimeric events (either from the same or different samples) were finally selected as candidates for the clustering of gene expression. LEUCEGENE data were downloaded from SRA using the fastq-dump utility (version 2.5.4) and converted to FASTQ. Using Kallisto 0.42.4 software and Ensembl 84 annotations, we determined transcript expression. Transcript counts computed by Kallisto were merged at gene-level19. The normalization of counts was performed with DESeq2 (version 1.14.1) (design ~ 1), so values used in the clustering were normalized counts transformed with the variance stabilization method provided in DESeq2 package. Heatmaps were produced with heatmap.2 function from the gplots package (R version number 3.3.2), using default parameters (i.e. complete-linkage clustering and Euclidean distance).\n\n\nResults\n\nWe performed RNA-seq on samples from 3 AML patients. One presented with a normal karyotype (NK), while the other two presented with an abnormal karyotype (AK), one with an Inv16 and the other with a t(15,17) translocation (sample names 1–3, Supplementary Table S1). The sequences were analyzed using Crac and CracTools. The selected chRNA candidates were tested by qPCR, and sequenced when qPCR displayed a positive signal. Some candidates could not be validated by qPCR due to the difficulty in designing suitable primers. The CBFB-MYH11 and PML-RAR fusion transcripts expressed in the AML-inv16 and AML-t(15;17) samples were identified using both RNA-seq and qPCR analysis, confirming the reliability of RNA-seq and the Crac suite in this type of analysis.\n\nWe identified 17 chRNAs among 44 candidates; 10/23 from AML-t(15,17), 4/18 from AML-NK, and 3/3 from AML-inv16 (Supplementary Table S2 and Supplementary Table S4). The validated chRNAs were distributed into 4 classes (definitions of these are in the materials and methods section) as follows:\n\n– Class 1: 5\n\n– Class 2: 3\n\n– Class 3: 5\n\n– Class 4: 4.\n\nAmong the Class 1 chRNAs more frequently associated with genomic translocation, we identified 4 chRNAs associated with PML and RAR genes in patient OM110223 suffering from AML-t(15,17). The remaining chRNA of this class was associated with the PAN3 gene and a non-annotated region (NONE), and was found in patient OS110089 suffering from AML-inv16. We highlighted two types of Class 2 chRNAs that depend on the genomic distance between the two parts of the read. A short distance was consistent with read-through, whereas a long distance would be associated with other mechanisms. We found two kinds of Class 3 chRNA - in the first, the chRNA processes the 3’ exon before the 5’ start of the same gene. In the second, the chimera involve distant exons from different genes. Finally we also validated several Class 4 chRNAs, among which the CBFB-MYH11 chimera associated with Inv16.\n\nA new Class 1 chRNA was identified in patient OS110089 presenting the inv16 associated fusion transcript CBFB-MYH11. The new fusion junction read (n° 14888 PAN3-NONE; Supplementary Table S2) corresponds to 13q12.2 and 2p21 chromosomal locations at the 5’ and 3’ of the RNA, respectively. The read chimeric annotation indicates a fusion between the 3’end of PAN3 exon18 (chr13+), with a non-annotated transcribed region “NONE” (chr2–pos 43193199-43193247) as shown in Figure 1A. The PAN3-NONE fusion transcript was validated by qPCR, and was only detected in patient OS110089 as shown in results of the AML cohort (Figure 5A). We compared its qPCR Ct value with the Ct value obtained with the amplification of the CBFB-MYH11 transcripts, and noticed that its value was higher (32 cycles vs 28, Supplementary Figure S3). This difference could be correlated either to a lower transcript expression level or to a heterogeneous expression in the tumor cell population. In order to determine whether this fusion transcript is associated with chromosomal rearrangement, FISH experiments were performed with a custom fusion probe. A corresponding translocation was observed in only 31% of the leukemia cells, demonstrating that the PAN3-NONE transcript belongs to a subclone, which could explain the lower expression level observed (Figure 1B). The analysis of the PAN3-NONE transcript during patient follow-up revealed its disappearance after the first induction of chemotherapy, without reappearance during the relapse period (Figure S3).\n\nA) Fusion junction description and primer design. The sequences of spanning junctions and paired reads supporting the chRNA and the corresponding designed primers are indicated. The orange and blue squares represent respectively the E17 and E18 of PAN3 gene (E16 of ENST00000399613.1 transcript), and the NONE part of the fusion transcript. The sequencing result of the PAN3-NONE SF1-SR1 PCR product is indicated. The symbol (#) denotes the part of the read which was not sequenced, () denotes the junction, and MCS indicates the multiple cloning sites used to clone the PCR product. (B) Translocation checking by FISH. The left panel (Control) represents normal blood cells and the right panel represents cells from patient OS110089. The presence of the NONE sequence on chr2 is denoted by the red signal and the presence of the PAN3 sequence on chr13 mentioned by the green signal. The right panel presents the NONE-PAN3 fusion at the chromosomal level (white box).\n\nWe next investigated the transcription of the non-annotated region “NONE” (chr2–pos 43193199-43193247) in normal and tumor tissues using the approach described previously combining digital Gene expression (DGE) and RNA-seq data20. Querying tissue expression profiles with DGE tags, we observed a tag in the NONE chromosomal area showing a specific expression in AML samples (Figure 2B). The RNA-seq read coverage and the DGE tag (Figure 2A) confirmed a new transcribed region, validated by qPCR in tumor cell lines (Figure 2C). We confirmed the presence of the NONE specific expression in AML and normal CD34+ hematopoietic stem cells (HSC) using a tag search approach in a largest RNA-seq collection of normal and tumor tissues (Table S5 and data not shown). Together, these results revealed a new lincRNA specifically expressed in hematopoietic lineage and most highly expressed in AML samples. Moreover, this lincRNA contributed to the formation of a new chRNA in the case of a translocation between chromosome 13 and 2.\n\nA) Display of the ENSEMBL genome browser viewer for the NONE transcribed region. The blue horizontal bars represent the genomic sequence. The histogram of the RNA-Seq coverage in the chromosomal region is displayed on both strands (OS110089 stranded and RNA-seq fwd/rev tracks). Public and personal DGE data (‘DGE tag location’ track: blue rectangle for occurrence>2) are displayed on both strands of the chromosome, with their relative occurrences (histogram of ‘DGE expression level’ track) using a private DAS server. (B) Relative expression of the NONE tag per million reads in the DGE datasets (Philippe, et al. 2013). (C) Relative expression of the NONE transcript in different cell lines and AML samples (NT refers to not treated cells). The relative gene expression was determined using the 2-ΔΔCT method. Transcriptional modulation was calculated by comparing various lineages with SH-SY5Y (subline of the neuroblastoma cell line SK-N-SH). For normalization, RPS19 was selected as a reference transcript. Standard deviation was measured using duplicate.\n\nFour Class 1 junctions involving PML and RARA genes were identified from the analysis of the AML-t(15; 17) OM110223 sample (Supplementary Table S2). Acute promyelocytic leukemia (APL) molecular diagnosis and minimal residual disease (MDR) monitoring are currently based on bcr1, bcr2 or bcr3 fusion transcript detection, depending on the DNA breakpoint21,22. PCR and FISH analyses performed during diagnosis showed that patient OM110223’s leukemia cells present a short bcr3 transcript which connects exon 3 of the PML gene and exon 9 of the RARA gene (equivalent to the exon 3 of the RARA-001 ENSEMBL transcript).\n\nAmong the 4 PML-RARA characterized junctions, one of them confirms the short bcr3 variant expression and the “reciprocal” transcript joining the RARA exon 5 and PML exon 423. Besides these fusion transcripts already described23,24, we observed the presence of two new fusion transcripts. The first corresponds to a fusion RNA shorter than bcr3, joining PML exon3 and RARA exon 12 (Figure 3A). This junction was detected in NB4 cells which express a bcr1 transcript as well as in ATRA treated cells from another bcr3 patient (OS000002 in Figure 3C). The corresponding protein lacks the RARA DNA binding domain present in previously described fusion proteins (Figure 3D). Moreover, different fusion transcripts linked to the translocation between the chr15 and 17 can coexist in a tumor sample, with their expression changing with time or treatment (Figure 3C). The second new fusion transcript corresponds to a chRNA that joined the exon 3 of a known RARA antisense transcript with the antisense part of PML intronic region (Figure 3B). The primers were designed in the corresponding PML intron free from known transcription, in order to amplify the antisense chRNA. This transcript was only detected in the sequenced sample. The sequencing results of PML-RARA chRNA qPCR amplifications revealed another fusion transcript joining PML exon 3 and RARA exon 10 present in OM110223 leukemia cells, without corresponding spanning junction read.\n\n(A) Scheme of PML and RARA genes are shown on the top, with the potential breakpoints. Below is the schematic of the chromosomal translocation, with visualization of the short bcr3 fusion transcript. Gene structures for both PML and RARA genes (ENSEMBL genome browser), and exons involved in new PML-RARA chimera are shown. We assigned a number to exons (E) of each gene. Bcr3 fusions found in this study are shown below, including the newly found fusion between exon 3 of PML and exon 12 of RARA. (B) New RARA-PML antisense fusion transcript. Schematic of RARA (blue) and PML (red) antisense transcript positions relative to their sense transcript, and the resulting antisense fusion transcript, are shown below. (C) Identification of selected PML-RARA transcript junctions in acute promyelocytic leukemia samples and NB4 cells by qPCR. (D) Hypothetical incidence of PML-RARA fusion transcript on protein domains. Sanger sequencing results of the 3-12 PML-RARA fusion is shown on the top. The hypothetical protein product is presented below, as well as the protein from the exon 3–9 fusion.\n\nFour classes of chimera were identified using the Crac and CracTools pipeline. Associated with specific features and annotations, subcategories could be defined and linked to potential biological mechanisms (Figure 4). Among the Class 2 chimera, we observed two distinct categories, depending on the vicinity of the two relevant genes (Figure 4A). The first category involves a junction between non-adjacent exons separated by thousands of base pairs (SLC16A3-METRNL and UBR5-AZIN1, Figure 4A and Table S2). The validated fusion transcript joins the SLC16A3 exon 2 with the METRNL start exon 5 at a distance of 855828bp. As shown from RNA-seq data, the region between the two genes is transcribed (data not shown), which calls into question the hypothesis of an intra-chromosomal deletion.\n\nThe position and orientation of the genes on the chromosomes have been schematically arranged. The start and end positions of the exons have been indicated. The representations of the chRNA junction supported by the Sanger sequencing results are also shown. (A) Two subtypes illustrating Class 2 chimera junctions. The first subtype1 is illustrated by the junction of two non-adjacent genes SLC16A3 and METRNL, separated by 855828 bp. The second subtype2 is illustrated by the junction between adjacent genes VAMP8 and VAMP5, separated by 2377 bp. (B) Three subtypes illustrating Class 3 chimera junction. They all involve the inversion of exon order, first two concern an inversion within a same exon of a gene (METRNL and FLT3 cases), and the third subtype involves an inversion of a transcribed sequence, separated by about 103 000 bp (NONE-CTDP1). (C) A Class 4 chimeric construction joining an exon from (+) strand gene TBCD22A with an exon from (-) strand gene CERK, arising from the same chromosome 22.\n\nThe second subcategory concerns two adjacent genes, fused with a loss of some exons. In the illustrated example of VAMP8-VAMP5, VAMP8-001 transcript exon 4 joined VAMP5-001 transcript exon 2 (Figure 4A). The subgroup most likely defines a read through transcript category with an alternative splicing.\n\nThe Class 3 chimera could reflect three different mechanisms associated with: genomic duplication, polymorphism, or intrinsic transcriptional mechanisms (Figure 4B). Two cases concern intra-exonic transcripts, with one exon end being present before the start of the same exon. This subcategory is illustrated by two fusion transcripts (METRNL; Figure 4B and RNF220 data not shown). In the transcript METRNL001, the 3’ part of exon 2 (chr17 (+)/pos 81043015-81043199) is present upstream of the 5’ part of exon 2 (chr17 (+)/pos 81042813-81042850). Such transcript could be generated either by transcriptional event or by the transcription of a rearranged allele. Indeed, this subgroup, intragenic chRNA, could highlight the presence of tandem duplication. As an example, we also identified the well-known FLT3 tandem repeat involved in acute myeloid leukemia. This type of tandem duplication is characterized by an overlap in the read’s genomic positioning, which can be easily detected using our workflow (Figure 4B).\n\nThe last case involves two distant genes. The genomic location of both transcripts is on the same chromosome but at a distance of 102781bp. We identified the chimera NONE-CTDP1 in the OM110223 patient sample. The transcript of a non-annotated chr18 (+)/pos 77557943-77558014 segment is fused with exon2-CTDP1-001 or with exon13-CTDP1-001 (Figure 4B).\n\nThe Class 4 chRNA corresponds to reads whose 5’ and 3’ parts match on the chromosome’s opposite strands (Figure 4C). This kind of chimera reflects a genomic inversion like CBFB-MYH11 in the absence of overlapping elements in the read. Besides the widely described inv16 (CBFB-MYH11), we validated three chRNA linked with a possible chromosomal inversion (TBC1D22A-CERK, MAEA-CTBP1, DHRS7B-TMEM11). In the TBCD22A-CERK fusion transcript, the TBCD22A exon 20 end is fused with the CERK exon 2 start (Figure 4C).\n\nTo extend our analysis, we tested the expression of the validated chRNA in a large cohort of AML samples with different karyotypes (Figure 5A, Table S1) using qPCR. We then classified the chRNA into AML subtypes or tumor-specific chRNA subgroups, taking into account the frequency and tissue-specific expression. We also distinguished the non-tumor chRNA by examining their expression in normal PBMCs. Among the 15 chRNA tested, some of them were widely expressed in all AML subtypes (RNF220-RNF220, METRNL-METRNL and MEA-CTDP1). The frequency and tissue-specific expression did not depend on the chRNA classes.\n\n(A) Expression of validated chRNA in a large AML cohort. ChRNA classes are identified by colours (green for Class 1, orange for Class 2, red for Class 3 and blue for Class 4). Karyotypes are indicated below the sample name (UK for unknown karyotype, NK for normal karyotype). For abnormal karyotype, chromosomal rearrangement is indicated. The screening was also performed on not treated (NT) or differentiated cell lines. For PML-RARA and CTDP1 exons involved in the chRNA are indicated. (B) Classification of chRNA depends on normal or tumor level expression. ChRNA expression, in normal CD34+ HSCs (CD34) and in AML (LEUCEGENE data), is presented using the tag search approach. For each group (AML-test, AML-AK, AML-NK, CD34), samples displaying a positive chRNA tag count are selected. The average tag expression is calculated with the selected samples and chRNA classified by their relative expression in the CD34 group. The table with highlighted colour indicates for each chimera, within the different cohort, the number of samples presenting the TAG.\n\nTo further extend our analysis and in order to validate our strategy on a large cohort, we analyzed a publicly available dataset of 125 AML and 17 normal CD34+ HSC RNA-seq using a tag search approach (see Materials and methods). For this purpose, we selected qPCR validated chRNA (Table S4) and candidates previously untested due to difficulty in designing primers (Table S2). The chRNAs were classified by their relative expression in normal CD34+ HSCs, in order to distinguish non-tumor and tumor-specific ones (Figure 5B). Among the chRNAs expressed in CD34+ HSCs, we observed different profiles: those more highly expressed in normal CD34+ cells than in AML, and those with low expression in CD34+ HSCs (see NSFL1-SIRPB2, METRNL-METRNL).\n\nWe identified four new types of tumor-specific chRNA; TRIM28-TRIM28, DHRS7B-TMEME11, PLXNB-BLRD1 and SLC16A3-METRNL, expressed in all AML groups (Figure 5B). DHRS7B-TMEM11 and PLXNB-BLRD1 transcripts are most abundant in the AML test group, whereas TRIM28-TRIM28 and SLC16A3-METRNL are equally expressed in the three AML groups (AML-test, 82 AML-AK, 43 AML-NK). It is worth noticing the involvement of the METRNL gene in two identified chimeras. TRIM28-TRIM28, FLT3-FLT3, PML-RARA (with (3–9) or (5–4) junction) and CBFB-MYH11 tag counts showed high mean expression levels (see Figure S4). FLT3, PML-RARA and CBFB-MYH11 are strong markers in AML, and also useful for prognostic and MDR monitoring. The FLT3 tag identified in OM110223 is not found in other samples, yet the pipeline reveals other FLT3 fusion transcripts, with different sequences in other AMLs (data not shown), indicating several variations at this fusion point. Among the new, TRIM28-TRIM28 chRNA is present at low frequency in normal and abnormal AML karyotype (2/43 AML-NK and 9/82 AML-AK). The high expression level of this chRNA in positive samples (comparable to previously described known markers CBFB-MYH11, FLT3-FLT3 and PML-RARA), suggests it could have a key role in such tumors.\n\nIn order to verify whether genes involved in chRNA have an aberrant expression profile25 that could influence tumorigenesis, we analyzed the impact of gene expression on AML with unsupervised clustering. As described for the read-through chRNA subcategory, we compared RNA-seq data from normal CD34+ HSC and AML-AK subtypes (LEUCEGENE, part3). Figure 6 shows a quantitative analysis obtained with “read-through related” genes of the input cohort. Known AK-AML subgroups could be distinguished from CD34+ HSC by their expression profile. We performed a similar study with the Class 3 tandem duplication subgroup, showing interesting differential profiles with “tandem duplication related” genes, mostly comprised of the newly identified TRIM28, CEBPD and FLT3 (Supplementary Figure S5).\n\nThe gene expression profile is analyzed by an unsupervised clustering method. Normal CD34+ HSC (CD34) and AML subgroups (LEUCEGENE RNA-seq data) were compared by analyzing the expression of a set of genes involved in read-through chRNAs. Parameters for gene selection are described in the Materials and methods section. Samples are identified by colours (black for normal CD34, pink for AML t(8;21), grey for AML t(9;11), green for AML-inv16). Gene names are indicated on the right.\n\n\nDiscussion\n\nThe use of RNA-seq to provide a detailed view of the transcriptome and to detect new RNA transcripts, opens up new opportunities for improving diagnosis and treatment of human disease. The characterization of new chRNA presents as a great opportunity, as it could reveal new transcriptomic biomarkers for cancer and therefore could be useful in personalized medicine. ChRNAs, also known as “fusion RNA” or “canonical chimeras”(5,26), are already used in diagnosis, but many other chimeric fusion products generated by transcriptional mechanisms such as read-throughs, cis or trans-splicing5,7,9, also have the potential to be used in diagnosis if correctly categorized. In this study, we successfully developed new tools to classify these fusion transcripts methodologically and biologically into chRNA categories and subcategories, and associated them with biological mechanisms.\n\nOne of the major challenges in chRNA detection is to distinguish true candidates from false positives during RNA-seq analysis. False positives can result from technical artefacts that occur during sample preparation, mainly produced by reverse transcription or downstream PCR errors27. Bioinformatics processes also generate artefacts associated with the algorithm’s approach for mapping raw reads to the complex reference genome. Many attempts have been made to improve the bioinformatics analysis of chRNAs by proposing multistep filtering pipelines including gene annotation, lists of known fusion genes or machine learning approaches to improve prediction13. However, pipeline choice remains a difficult task for biologists and bioinformaticians. We have developed a benchmarking system that enables the calibration and selection of pipelines optimised for the detection of fusion RNAs. Our work also entailed developing, within Crac, a machine learning model used to optimise the selection of fusion RNAs (personal data, submitted for publication). Crac offers precise prediction of all chRNA categories, and the CracTools pipeline helps the biologist increase biological rate validation, producing a ChimValue that takes into account methodology and annotation.\n\nAs mentioned above, what is at stake is the possibility of exploring the “whole chimeric transcriptome” to classify chRNA, and hence identify cancer biomarkers. High throughput studies, like the Cancer Genome Project6,28,29, performed on large cohorts with thousands of experiments, have focused on “canonical fusion genes”. Most of the molecular cancer markers are based on fusion genes because of their ease of detection. It is more difficult to identify features that distinguish chRNAs into new categories, and to elucidate their biological mechanisms and functions. Integrating DNA-seq and RNA-seq data to improve classification is a possible solution5,30, but it is time and cost consuming. In this work, we confirm that RNA-seq is a good solution for canonical and new chRNA extraction, and propose a classification system based on subcategories and specific expression profiles.\n\nThe present study also reveals new chRNA candidates among the well characterized subcategories. We identified a Class 1 PAN3-NONE chRNA transcript associated with a new translocation in a tumor subclone of a characterized Inv(16) AML, that could be used in patient follow-up. We also identified novel PML-RAR isoforms, shorter than the isoforms currently used in diagnosis, which could again be used in patient follow-up. A recent publication revealed that several isoforms can coexist in leukemia cells from the same patient. The authors showed that the ATRA cell response is isoform-dependent, as the short isoform lacks sensitivity to ATRA31. MRD and patient follow-up in APL is usually performed by PML-RAR transcript QPCR, and relapse is associated with an increase of bcr1, bcr2 or bcr3 fusion transcripts32. Then, it would be useful to have a picture of the complete isoforms, to best address treatment in this context. Though many patients with AML-inv16 or AML-t(15;17) can benefit from effective treatment, some may develop resistance, leading to adverse outcomes. The appearance or increase of fusion transcripts during treatment could be an indicator of such resistance.\n\nBesides the translocation and inversion mechanisms, our pipeline highlights other events that correlate with chromosomal rearrangement and cancer diagnosis and prognosis. We find Class 3 overlap fusion transcripts like FLT3, corresponding to tandem duplication that could be of use in the prognosis and MRD of AML33. The real advantages of RNA-seq in highlighting tandem repeat sequences are its open nature and its capacity to detect outside “hotspots”. Furthermore, we also detected fusion transcripts resulting from “read-through transcription”, described in chRNA studies on cancer.\n\nMost newly identified chRNAs used canonical splice sites and were detected in normal hematopoietic tissues. This observation confirms previously published works concerning the recurrence of chimeric fusion RNAs in healthy cells34. It is unlikely that they are the products of genomic abnormalities, since they are expressed in healthy samples. However, the pathogenetic impact of these chimeric fusions remains unclear. Recent findings have demonstrated the role of read-through chRNA in renal carcinoma and breast cancer26,35, and our data demonstrates that genes involved in these events are differently expressed in AML. More studies are needed to elucidate the physiopathological impact of these chRNAs.\n\nThe potential of NGS technologies, particularly of RNA-seq, in increasing the capabilities of personalized medicine is clear2. However, to achieve this, efforts must be made to facilitate the interpretation of complex high throughput data. For chRNA, this is feasible only if the fusion transcripts are well classified and characterized. New technologies are available to simplify disease follow-up at reduced costs. Here, we propose a robust, open method based on a single process to identify different classes of chRNA. This approach provides a chRNA transcriptome map of biomarkers for disease characterization and monitoring including known canonical gene fusions and new chRNA. In combination with a tag-based approach and gene expression profile, this map can give a global picture of the complex physiological processes and could correlate with current leukemia classification.\n\nAbbreviations: chRNA, chimeric RNA; AML, acute myeloid leukemia; NK, normal karyotype; UK, unknown karyotype; AK, abnormal karyotype; APL, Acute Promyelocytic Leukemia; PBMCs, peripheral blood mononuclear cells; Inv16, chromosome 16 inversion; t(15;17), translocation of chromosomes 15 and 17; qPCR, quantitative polymerase chain reaction; NONE, non-annotated region; LincRNA, Long intergenic noncoding RNAs; Bcr, break chromosomal region; FISH, Fluorescence in situ hybridization; ATRA, all-trans retinoic acid; VD, vitamin D; TGFβtransforming growth factor beta; MRD, minimal residual disease.\n\n\nData availability\n\nThe Crac and CracTools software is hosted on http://crac.gforge.inria.fr/.\n\nRaw data (FASTQ files) for OM100011, OM110223 and OS110089 patients are available under accession number E-MTAB-5767 in the ArrayExpress database at EBI.\n\nThe publicly available ENCODE datasets (a detailed list is provided in Supplementary Table S7) used in the study are available on https://www.ENCODEproject.org/.\n\nThe following LEUCEGENE datasets: GSE62190 (82AML-AK), GSE48846 (17 CD34), GSE49642 (43AML-NK) (a detailed list is provided in Supplementary Table S6) are available on https://www.ncbi.nlm.nih.gov/geo/\n\nGRCh37/Hg19 genome sequences are available at ftp://ftp.ensembl.org/pub/grch37/release-88/fasta/homo_sapiens/dna/.\n\nThe annotations GFF file from the ENSEMBL genome browser is available at ftp://ftp.ensembl.org/pub/grch37/release-84/gff3/homo_sapiens/Homo_sapiens.GRCh37.82.gff3.gz.\n\n\nConsent\n\nThe patients and healthy donors provided written informed consent to participate in the study, in accordance with the Declaration of Helsinki. The study was approved by the ethics board of Nîmes University (CCPPRB 2002/1103).", "appendix": "Author contributions\n\n\n\nFR, NP and TC initiated the project. JA and NP developed the pipelines. JA, AB and SB performed the analytic work. JA performed clustering analysis. JBG performed fish analysis. EBS, SB and FR performed biological validation. AM, BC, CC provided biological samples, diagnostic tests and specific help for chRNA validation. RA, SR, performed tag RNA-seq data sets selection and tag search approach. NG performed control RT experiments. JML and ALB contributed to scientific discussion. FR, TC wrote the paper. ALB contributed to manuscript revisions. All authors read and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the French ANR IBC project “Investissement d’avenir en bioinformatique-projet-IBC” and FRM “Appel d’offres urgence pour la bioinformatique, projet DBI20131228566”. This work was supported by the France Génomique National infrastructure, funded as part of the “Investissements d’Avenir” program managed by the Agence Nationale pour la Recherche (ANR-10-INBS-09). We acknowledge la Ligue Nationale Contre le Cancer for financial support (EL2015.LNCC/JML) to JML’s team.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nJérôme Audoux is fellowship of the FRM (Appel d’offres urgence pour la bioinformatique, projet DBI20131228566) and Ronnie Alves are fellowships of the IBC (ANR “Investissement d’avenir en bioinformatique-projet-IBC); We thank Philippe Clair (Engineer, University of Montpellier, France) for advice on qPCR (qPHD plateform, Montpellier GenomiX) and Jean-Marc Holder (CMO SeqOne) for text corrections.\n\n\nSupplementary material\n\nFigure S1: PCR strategy\n\nPCR primers were designed on both sides of the junction point (*) using spanning junction read or a sequence reconstructed by combining paired-end and chimeric spanning reads (meta-read). SF and SR indicated respectively short forward and short reverse primers, LF and LR, long forward and long reverse primers. Forward (F) and reverse (R) indicated primers pairs designed on spanning meta-reads.\n\nClick here to access the data.\n\nFigure S2: NONE transcript amplification strategy\n\nThe PAN3-NONE read sequence and its corresponding genomic position on GrCH37 is shown. The junction point indicated the chr2: 43193247 (-) positions for NONE transcript. From this position, we selected an upstream 100 base pairs sequence in order to amplify the putative NONE transcript. Primers were designed on each part of the chr2: 43193247 position to specifically amplify the non-chimeric transcript.\n\nClick here to access the data.\n\nFigure S3: PAN3-NONE qPCR amplification\n\nBoth PAN3-NONE and CBFB-MYH11 chRNAs were amplified from each sample. CBFB-MYH11 chRNA, was used as a positive control. For each amplicon, the Ct value and the melting temperature (Tm) are shown (upper table).\n\nThe second table shows PAN3-NONE and CBFB-MYH11 qPCR results for the time points corresponding to the 2 years follow-up of the OS110089 patient. Clinical information and QPRC results for both fusion transcripts are mentioned. The molecular biology for CBFB-MYH11 (Inv16) performed by the diagnostic laboratory is also indicated as positive or percentage values; DNQ= detected-not quantified; ND= not detected.\n\nClick here to access the data.\n\nFigure S4: Expression of ChRNA in different AML groups and hematopoietic cells by tag counting approach.\n\nFor each group (AML-test, AML-AK, AML-NK and CD34), the average Tags mean expression is indicated. Tags were designed for the indicated chRNA as described in materials and methods. Only positive samples were retained to calculate the average tag expression. The tag counts are normalized per 5 billion of total k-mers.\n\nClick here to access the data.\n\nFigure S5: Expression profile of genes involved in Class3 tandem repeat chRNAs.\n\nHeat map representing colour-coded expression levels of class3 chimeras (chRNAs normalized count) across patient samples. Names of genes involved in class3 chRNAs are indicated on the right. Black, pink, green and grey bars at the top represent respectively normal CD34, AML-t(8;21), AML-inv16, AML-t(9;11).\n\nClick here to access the data.\n\nTable S1: Patient data.\n\nID and patient information for the AML cohort.\n\nClick here to access the data.\n\nTable S2: CRAC & Cractools pipeline output obtained from OS110089, OM100011 and OM110223 RNAseq data analysis.\n\nClick here to access the data.\n\nTable S3: Primers table for chRNA qPCR validation.\n\nClick here to access the data.\n\nTable S4: Validated chRNAs information\n\nThe table gives the list of TAGs used for tag search approach. For the chRNA, exons involved in the chimeric junction and tag sequences used for the tag search approach are indicated.\n\nClick here to access the data.\n\nTable S5: NONE and PAN3 tags expression within a set of libraries\n\nSpecific tags for PAN3 and non-chimeric NONE transcript corresponding respectively to Chr13(+) : 28813770-28813799 and Chr2(-) : 43193287-43193316 position on GRCh37/Hg19 genome were designed and counted in FASTQ files of 52 libraries from various tissues. Expression was normalized per 5 billion of total k-mers.\n\nClick here to access the data.\n\nTable S6: List of LEUCEGENE and ENCODE datasets used in the study\n\nTable S6 describes the different parts of the LEUCEGENE cohort used in the study. Samples indicated by a GSM number are classified by groups of different Karyoptype. Table S6 also indicates the ENCODE dataset used in the study.\n\nClick here to access the data.\n\n\nReferences\n\nMaher CA, Palanisamy N, Brenner JC, et al.: Chimeric transcript discovery by paired-end transcriptome sequencing. Proc Natl Acad Sci U S A. 2009; 106(30): 12353–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nByron SA, Van Keuren-Jensen KR, Engelthaler DM, et al.: Translating RNA sequencing into clinical diagnostics: opportunities and challenges. Nat Rev Genet. 2016; 17(5): 257–71. PubMed Abstract \| Publisher Full Text\n\nAtlas of Genetics and Cytogenetics in Oncology and Haematology [Internet]. [cité 28 janv 2017]. Reference Source\n\nMitelman Database of Chromosome Aberrations and Gene Fusions in Cancer [Internet]. [cité 28 janv 2017]. Reference Source\n\nMertens F, Johansson B, Fioretos T, et al.: The emerging complexity of gene fusions in cancer. Nat Rev Cancer. 2015; 15(6): 371–81. 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PubMed Abstract \| Publisher Full Text\n\nPhilippe N, Bou Samra E, Boureux A, et al.: Combining DGE and RNA-sequencing data to identify new polyA+ non-coding transcripts in the human genome. Nucleic Acids Res. 2014; 42(5): 2820–32. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nvan Dongen JJ, Macintyre EA, Gabert JA, et al.: Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia. 1999; 13(12): 1901–28. PubMed Abstract\n\nGabert J, Beillard E, van der Velden VH, et al.: Standardization and quality control studies of ‘real-time’ quantitative reverse transcriptase polymerase chain reaction of fusion gene transcripts for residual disease detection in leukemia – A Europe Against Cancer Program. Leukemia. 2003; 17(12): 2318–57. PubMed Abstract \| Publisher Full Text\n\nWalz C, Grimwade D, Saussele S, et al.: Atypical mRNA fusions in PML-RARA positive, RARA-PML negative acute promyelocytic leukemia. Genes Chromosomes Cancer. 2010; 49(5): 471–9. PubMed Abstract \| Publisher Full Text\n\nPandolfi PP, Alcalay M, Fagioli M, et al.: Genomic variability and alternative splicing generate multiple PML/RAR alpha transcripts that ENCODE aberrant PML proteins and PML/RAR alpha isoforms in acute promyelocytic leukaemia. EMBO J. 1992; 11(4): 1397–407. PubMed Abstract \| Free Full Text\n\nGrosso AR, Leite AP, Carvalho S, et al.: Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma. eLife. 2015; 4: pii: e09214. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJividen K, Li H: Chimeric RNAs generated by intergenic splicing in normal and cancer cells. Genes Chromosomes Cancer. 2014; 53(12): 963–71. PubMed Abstract \| Publisher Full Text\n\nPeng Z, Yuan C, Zellmer L, et al.: Hypothesis: Artifacts, Including Spurious Chimeric RNAs with a Short Homologous Sequence, Caused by Consecutive Reverse Transcriptions and Endogenous Random Primers. J Cancer. 2015; 6(6): 555–67. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nInternational Cancer Genome Consortium, Hudson TJ, Anderson W, et al.: International network of cancer genome projects. Nature. 2010; 464(7291): 993–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCancer Genome Atlas Research Network, Ley TJ, Miller C, et al.: Genomic and Epigenomic Landscapes of Adult De Novo Acute Myeloid Leukemia. N Engl J Med. 2013; 368(22): 2059–74. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhang J, White NM, Schmidt HK, et al.: INTEGRATE: Gene fusion discovery using whole genome and transcriptome data. Genome Res. 2016; 26(1): 108–18. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan Y, Bian S, Xu Z, et al.: The short isoform of the long-type PML-RARA fusion gene in acute promyelocytic leukaemia lacks sensitivity to all-trans-retinoic acid. Br J Haematol. 2013; 162(1): 93–7. PubMed Abstract \| Publisher Full Text\n\nCassinat B, de Botton S, Kelaidi C, et al.: When can real-time quantitative RT-PCR effectively define molecular relapse in acute promyelocytic leukemia patients? (Results of the French Belgian Swiss APL Group). Leuk Res. 2009; 33(9): 1178–82. PubMed Abstract \| Publisher Full Text\n\nBibault JE, Figeac M, Hélevaut N, et al.: Next-generation sequencing of FLT3 internal tandem duplications for minimal residual disease monitoring in acute myeloid leukemia. Oncotarget. 2015; 6(26): 22812–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBabiceanu M, Qin F, Xie Z, et al.: Recurrent chimeric fusion RNAs in non-cancer tissues and cells. Nucl Acids Res. 2016; 44(6): 2859–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVarley KE, Gertz J, Roberts BS, et al.: Recurrent read-through fusion transcripts in breast cancer. Breast Cancer Res Treat. 2014; 146(2): 287–97. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "25101", "date": "29 Aug 2017", "name": "Hui Li", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRuffle et al., performed RNA-Seq on three AML patients, and identified a number of new chimeric RNAs. They grouped them into reasonable categories, and discussed their generating mechanisms. For some, they performed tag search in larger sets of RNA-Seq data of AML and CD34+ HSCs. In general, the analysis is solid. Multiple levels of evidence were provided for some fusion RNAs (for instance FISH for a Class I fusion). The conclusion is justified.\nNo major issues were noted.\nMinor suggestions:\nIt would be nice to perform the same RNA-Seq pipeline analysis on bigger datasets, such as the data from LEUCEGENE.\n\nIt is well known that different software tools behave differently and false positive/negative is a big issue. Would be nice to use another independent software for crosschecking.\n\nThe METRNL001 Class 3 chimera may be a product of backsplicing if the sequencing protocol is not restricted to polyA RNAs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3189", "date": "17 Nov 2017", "name": "Therese Commes", "role": "Author Response", "response": "Response to referee Report ( Hui LI, Department of Pathology, School of Medicine, University of Virginia, Charlottesville, VA, USA)1. It would be nice to perform the same RNAseq pipeline analysis on bigger datasets, such as the data from leucegene.2. It is well known that different software tools behave differently and false positive /negative is a big issue. Would be nice to use another independent software for crosschecking.3. The METRNL001 class3 chimera may be a product of backsplicing if the sequencing protocol is not restricted to polyA RNAsWe want to thank the referee for taking time to read the manuscript and provide constructive feedback.1. The first remark concerning LEUCEGENE analysis is relevant as this cohort is a unique dataset of 450 RNA-seq from AML patients and normal hematopoietic cells. This cohort includes diverse types of AML and clinical data with a deepness of 100-300x106 reads/per sample. However, our first aim in the present manuscript was to test our pipeline and to propose a new way to analyze, select and classify chRNA. We thus choose to analyze a small dataset and used the Leucegene cohort only to explore specific expression of the new chRNAs (revealed with our pipeline). This Expression with a tag search approach was based on their recurrence, tumor, subgroup, or patient-specific expression. The complete leucegene chRNA research represents a big task and constitutes a full-fledged work, we are now performing our RNAseq pipeline on this dataset. In this new task, we will check at a large scale the pipeline performance, in term of memory computing and time consuming, and also validate its capacity to detect all the well-known AML fusion genes. We then should be able to discover new chimeric RNA in AML. 2. We totally agree with the referee and are well aware of potential bias generated with different pipelines. Our group is particularly concerned about software comparison for chimeric RNA detection. CRAC was developed in the Lab and compared with other software before we choose it for this study (Philippe et al, 2013, Genome Biology; an integrated approach to the analysis of RNA-seq reads genome Biology) and we recently published in September 2017 a benchmark method dealing with this topic (Audoux et al, 2017, BMC Bioinformatics; SimBA: A methodology and tools for evaluating the performance of RNA-Seq bioinformatic pipelines). CRAC tools has been developed in the Lab in response to biologists requests to better characterize, filter and classify predicted fusion transcripts. Moreover, we recently compared the pipeline CRAC and CRACtools with Fusioncatcher in our AML dataset, as it provides interesting and complementary annotation (overlapping databases, protein impact) but the process is time consuming and the filtering step is more stringent, many of the new fusion transcripts we validated are ruled out.Whatever the bioinformatics pipeline used, bias will subsist. In order to improve chRNA discovery, we undertook to couple the bioinformatics approach with biological information and propose a tag search approach targeting the chimeric junctions in normal and tumoral large data set. We demonstrate that this complementary approach works well and will help the biologist to determine the biological relevance of chimeric events.3. Since we use polyA+ RNA to perform RNAseq, the METRNL001 class3 chimera, which involves a single exon (end of exon 2 METRNL transcribed before the start of the same exon) more probably corresponds to a linear polyA+ transcript involving a short repeat sequence of 5’ part of exon 2 as described in Fig4 rather to a circularization of a single exon formed by backsplicing." } ] }, { "id": "25306", "date": "03 Oct 2017", "name": "Charles Gawad", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this report, Ruffle et al. present a new approach for identifying novel chimeric transcripts using CracTools. They classify the transcripts into four categories: 1) different chromosomes (interchromosomal translocations), 2) co-linear but from different genes (putative transcriptional read through or complex intrachromosomal structural variation), 3) exons from same chromosome but are not in the expected order based on the reference (tandem duplication, complex intrachromosomal rearrangement), or 4) exons on same chromosome but different strands. The authors then go on to validate a subset of putative new chimeric transcripts using RT-PCR and FISH. These types of studies have been previously performed. One new aspect of this study is the stranded library preparation which allows for the identification of the class 4 chimeras. In addition, they tried to minimize the bias in their analysis pipeline by not relying on a reference genome, which enabled the discovery of new transcripts. Overall, their approach provides a validated new strategy for identifying novel transcripts in RNA-seq data.\nMajor Concerns\nThe authors do not discuss circular RNA, which are likely to make up a large portion of their class 3 chimeras as found in many recent studies. The low numbers of class 3 chimeras also raises concerns about the sensitivity of the approach, as most recent studies have found thousands of circular RNA isoforms per sample. The total RNA underwent RT with random primers, which would retain circular RNA. It is not clear if there was a polyA-selection step after that point or if the total RNA was ribosomal depleted. If it was the former, the circular transcripts would not be present.\n\nIf the RNA was not polyA-selected, the authors should specifically discuss the PML-RARA circular transcripts recently discovered in acute promyelocytic leukemia. I am not aware of any independent validation of that work.\n\nMinor Concerns\nThe authors should read the manuscript closely for typos. For example in the AML samples and cells lines section there are commas where there should be periods, in the FISH methods section there are degree signs instead of percent, and two paragraphs before the discussion there is MDR instead of MRD.\n\nThe authors should include the kits used for ribosomal RNA-depletion/polyA-selection, as well as stranded library preparation.\n\nWhat mechanisms do the authors have in mind for a structural variant and/or alternative splicing event that would result in class 4 chimeras, as they would not occur as a result of transcriptional read through of the same strand?\n\nThe resolution is too low for Figure 5A.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3229", "date": "19 Dec 2017", "name": "Therese Commes", "role": "Author Response", "response": "We thank the referee for his careful reading of our manuscript and for his remarks which led us to clarify the protocol used in this work. Major Concerns The authors do not discuss circular RNA, which are likely to make up a large portion of their class 3 chimeras as found in many recent studies. The low numbers of class 3 chimeras also raises concerns about the sensitivity of the approach, as most recent studies have found thousands of circular RNA isoforms per sample. The total RNA underwent RT with random primers, which would retain circular RNA. It is not clear if there was a polyA-selection step after that point or if the total RNA was ribosomal depleted. If it was the former, the circular transcripts would not be present. Response: The remark is relevant and we agree with the referee that it was not clearly stated that we performed polyA selection for the RNAseq experiment. This point is only described in online data availability (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5767/) and doesn’t appear explicitly in mat&methods section. We added the following comment on page 4: “The RNAseq was performed using polyA-selection with the TruSeq RNA Lib-Prep Kit (Illumina) adjusted with GATC specific procedure for strand specificity”. We also agree with the referee that class3 fusion transcripts could arise from circular RNA (circRNA) when performing ribosomal depleted RNA-seq. However, as our polyA+ RNA-seq study doesn’t enable the identification of this RNA subtype, we do not discuss about circRNA in the manuscript. Concerning class3 chimeric RNA, our pipeline detects a great number of candidates before applying specific filters. This category, as well as the class2, is the most represented in all datasets we analyzed. As described in mat&methods section, we used stringent criteria to minimize bias and greatly reduced the number of candidates. For example, from a typical AML stranded and paired- end RNA-seq experiment (50 Millions reads; 100pb length), starting from the raw data (CRAC and CracTools process) we extracted 1406 chRNAs including 35% of class3chRNA reduced by 16 fold with the filtering process (26 class3chRNA). If the RNA was not polyA-selected, the authors should specifically discuss the PML-RARA circular transcripts recently discovered in acute promyelocytic leukemia. I am not aware of any independent validation of that work. Response: As we choose polyA RNA-seq, we can only characterize new linear fusion transcripts. From ribo-depleted RNAseq data analysis, the characterization of circRNA arising from fusion gene requires the identification of linear junction (lin-J) and circular junction (circ-J) described as spliced and back spliced junction respectively by Guarnerio et al (Oncogenic Role of Fusion-circRNAs Derived from Cancer-Associated Chromosomal Translocations, 2016; Cell 165 (2): 289-302). The linear and circular fusion transcripts share the same linear junction. The new (3-12) PML-RARA junction we discover corresponds necessarily to a linear junction because of its fusion junction sequence (see figure at https://bio2m.montp.inserm.fr/papers/AMLchimera2017). Moreover, in our polyA+ dataset it surely corresponds to a linear transcript. It is most probably an alternative splicing product transcribed from the PML-RARA bcr3 translocation since it coexists with the well-known (3-9) PML-RARA bcr3 transcript. However we could not exclude in the biological sample the presence of circRNA emerging from the PML-RARA fusion gene. However polyA RNAseq protocol doesn’t enable to reveal them. To answer to the referee remark, we performed complementary experiments and analyzed the Guarnerio dataset (BioProjectID: PRJNA315254) with our pipeline to detect fusion junctions (lin-J and Circ-J) specific to circRNA arising from the PML-RARA translocated genes. We found 3 PML-RARA and 1 RARA-PML linear fusion junctions but we did not detect the circRNA ones. We performed a tag search approach specific to the PML-RARA circ-junction (F-circ1) described in Fig S1B supplemental information of Guarnerio et al manuscript. We did not find it in their fastQ files. To conclude we were unable to detect PML-RARA cirRNA in Guarnerio J dataset but we confirmed the data recently described by You, X. and Conrad, T. OF with the specific cirRNA Acfs pipeline (Acfs: accurate circRNA identification and quantification from RNA-Seq data, Scientific Report 6,38820; doi: 10.1038/srep38820; 2016). The circRNA search for low abundance will certainly require more deepness in RNAseq. Minor Concerns The authors should read the manuscript closely for typos. For example in the AML samples and cells lines section there are commas where there should be periods, in the FISH methods section there are degree signs instead of percent, and two paragraphs before the discussion there is MDR instead of MRD. Response: We read carefully the manuscript and corrected the typos. The authors should include the kits used for ribosomal RNA-depletion/polyA-selection, as well as stranded library preparation. Response: We added the following comment on page 4: “The RNAseq was performed using polyA-selection with the TruSeq RNA Lib-Prep Kit (Illumina, San Diego, CA) adjusted with GATC specific procedure for strand specificity”. What mechanisms do the authors have in mind for a structural variant and/or alternative splicing event that would result in class 4 chimeras, as they would not occur as a result of transcriptional read through of the same strand ? Response: Two mechanisms would result in class 4 chimeras. The first one could involve chromosomic duplication and inversion as described in Newman et al (Next-generation sequencing of duplication CNVs reveals that most are tandem and some create fusion genes at breakpoints, Am J Hum Genet. 2015, Feb 5; 96(2):208-20. doi:10.1016/j.ajhg. 2014.12.017. Epub 2015 Jan 29). The second one could involve splicing events as described by Gingeras et al (Implications of chimaeric non-co-linear transcripts, Nature. 2009 Sep 10; 461(7261):206-11doi: 10.1038/nature08452.) The resolution is too low for Figure 5A. Response: We changed the resolution of the figure 5A, increasing the font size." } ] } ]	1	https://f1000research.com/articles/6-1302
https://f1000research.com/articles/6-1950/v1	03 Nov 17	{ "type": "Research Note", "title": "Unexpected results in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity", "authors": [ "Gabriela Bautista", "Bence Mátyás", "Bence Mátyás" ], "abstract": "The number of studies investigating the effect of bio-fertilizers is increasing because of their importance in sustainable agriculture and environmental quality. In our experiments, we measured the effect of different fertilizers on soil respiration. In the present study, we were looking for the cause of unexpected changes in CO2 values while examining Chernozem soil samples. We concluded that CO2 oxidizing microbes or methanotrophs may be present in the soil that periodically consume CO2 . This is unusual for a sample taken from the upper layer of well-ventilated Chernozem soil with optimal moisture content.", "keywords": [ "bio-fertilizer", "soil respiration", "Chernozem", "OxiTop" ], "content": "Introduction\n\nResearch related to the benefits of microbes as biofertilizer has become increasingly important in the agricultural sector. This is due to their potential role in achieving higher crop yields while minimizing negative impact on the environment. It is well known that bio-fertilizers increase plant yield and improve soil fertility1–3. Soil respiration is an important indicator of soil microbial activity4–6. In our experiments, we measured the effect of different chemicals7–9 and a bio fertilizer on soil microbial activity, using both well-established and novel methods under laboratory conditions. We present some unexpected results from a setup in which Chernozem soil samples were examined.\n\n\nMethods\n\nThe phylazonit bio-fertilizer (produced by Phylazonit.Ltd, Hungary) was used for testing (15 l/ha). It has the following composition: Bacillus megaterium, Bacillus circulans, Pseudomonas putida, in an optimized ratio for soil injection. Number of bacteria: 109 piece/cm3.\n\nA total of 24 soil samples were collected near Debrecen, Hungary on the 19th April 2016, from an upper layer (0–20 cm) of Chernozem soil (47° 33’ 55.36” N; 21° 28’ 12.27” E). All samples had optimum moisture content with 19–21 percent. Soil moisture content was determined gravimetrically by drying the soil at 105 degrees C for 24 hours according to Klimes-Szmik, 197010. The experimental design was completely randomized, treatments were applied at 25 °C. An OxiTop OC110 respirometer was used to quantify the release and capture of CO2, automatically determined by the device after the biological oxygen demand (BOD) required for the degradation of organic matter has been measured. We used a 500 ml glass bottle system, following the protocol in the manual (https://www.wtw.com/en/service/downloads/operating-manuals.html). 10g of soil sample were placed into OxiTop flasks, capped with the sensor heads according to Barrales-Brito et al., 201411. 2.5 g of CO2 absorber (sodalime) were then added to a tank, to absorb the generated CO2 according to Barrales-Brito et al., 201411. Induced samples were given 0.1g of glucose . Each treatment was replicated four times. As Figure 1 shows, four samples were always measured in parallel: Absolute control (does not contain fertilizer, nor added glucose), Induced control (contains added glucose), Treated (contains bio-fertilizer) and Induced treated (contains bio-fertilizer and glucose). The Oxitop automatically provides the values related to CO2 production.\n\nThe induced method was also applied in order to differentiate between the control and the treated soil samples to become detectable sooner. Glucose was applied as inducer. As expected the CO2 values increase or stagnate.\n\n\nResults\n\nThe treated samples produced more CO2 than the controls, as expected. Each repeat with the exception of one showed growing CO2 values (Figure 1), as the pressure continuously decreased in the bottle due to gas (oxygen) consumption. One sample produced unexpected results (Figure 2). In the first 12 hours, the treated samples produced more CO2 than the controls in each measurement. Following this, a fluctuation in the values was observed.\n\nAfter examining the Oxitop device’s operation, this pattern became more interesting for us, as the device quantifies CO2 production by measuring BOD required for the degradation of organic matter. From the decreasing CO2 values, we conclude that there was oxygen production and/or CO2 consumption in the Oxitop bottles.\n\n\nDiscussion\n\nIn a closed system where the pressure decreases due to oxygen consumption, the values must increase or stagnate with the passage of time, but this was not the case with one of the samples (Figure 2). Here, a decrease in CO2 occurred. The following possible reasons were excluded:\n\nPresence of algae: there was no light in the incubator, so there was no photosynthesis.\n\nChanging pressure caused by changing temperature: the temperature was constant in the setup.\n\nAbsorption by the water in the sample: all other samples that produced increasing amount of CO2 had the same or comparable moisture content.\n\nOne reason that seemed more likely was that CO2 oxidizing microbes or methanotrophs may have been present in the soil, periodically using the produced CO2. This is unusual since most of studies report the presence of these bacteria in seawater12, paddy fields13 or industrial processes14 and not in well-ventilated Chernozem soil. Further genomics research could detect the bacterial strains that consumed the CO2 in this soil.\n\n\nData availability\n\nDataset 1: Average values for a number of different soil properties. DOI, 10.5256/f1000research.12936.d18265515.\n\nDataset 2: Average values of produced CO2 (ml/l) with different treatments. ’Control’ does not contain fertilizer, nor added glucose. ’Control+Glucose’ contains 0,1 g of added glucose. ’Biofertilizer’ contains Phylazonit bio-fertilizer. ’Biofertilizer+Glucose’ contains Phylazonit bio-fertilizer and 0,1 g of added glucose. DOI, 10.5256/f1000research.12936.d18266316.\n\nDataset 3: Comparison of produced CO2 (ml/l) in the sample in which unexpected (periodically decreasing CO2) values can be observed. ’Control’ does not contain fertilizer, nor added glucose. ’Control+Glucose’ contains 0,1 g of added glucose. ’Biofertilizer’ contains Phylazonit bio-fertilizer. ’Biofertilizer+Glucose’ contains Phylazonit bio-fertilizer and 0,1 g of added glucose. DOI, 10.5256/f1000research.12936.d18266417.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to the Department of Agricultural Chemistry and Soil Sciences at University of Debrecen for providing the experimental setups.\n\n\nReferences\n\nEl-Yazeid A, Abou-Aly H, Mady M, et al.: Enhancing growth, productivity and quality of squash plants using phosphate dissolving microorganisms (bio phosphor) combined with boron foliar spray. Res J Agr Biol Sci. 2007; 3(4): 274–286. Reference Source\n\nGarg V, Kukreja K, Singla A: Efficacy of native rhizobial strains for nodulation and plant biomass production in berseem (Trifolium alexandrinum L). AgricInternational. 2016; 3(1–2): 89–95. Reference Source\n\nGarg V, Kukreja K, Gera R, et al.: Production of indole-3-acetic acid by berseem (Trifolium alexandrinum L.) rhizobia isolated from Haryana, India. Agr Sci Digest. 2015; 35(3): 229–232. Publisher Full Text\n\nAnderson TH, Domsch KH: The metabolic quotient for CO2 (qCO2) as a specific activity parameter to assess the effects of environmental conditions, such as ph, on the microbial biomass of forest soils. Soil Biol Biochem. 1993; 25(3): 393–395. Publisher Full Text\n\nAllison SD, Czimczik CI, Treseder KK: Microbial activity and soil respiration under nitrogen addition in Alaskan boreal forest. Glob Chang Biol. 2008; 14(5): 1156–1168. Publisher Full Text\n\nCachipuendo-Ulcuango CJ, Requelme N, Gualavisí Cachiguango OM, et al.: Community use of water and land for sustainable production of pasture. La Granja: Journal of Life Sciences. 2017; 26(2): 142–154. Publisher Full Text\n\nMátyás B, Kátai J, Horváth J: Comparative analysis of certain soil microbiological characteristics of carbon cycle. Acta Agraria. 2016; (69): 137–142. Reference Source\n\nHorváth J, Mátyás B, Kátai J: Impact of agronomic factors on urease enzyme activity in a long term fertilizer experiment. Acta Agraria. 2016; (68): 43–48. Reference Source\n\nMátyás B, Tállai M, Kátai J, et al.: The impact of fertilisation on a few microbiological parameters of the carbon cycle. Acta Agraria. 2015; (64): 45–50. Reference Source\n\nKlimes-Szmik A: A talajok fizikai tulajdonsá-gainak vizsgálata. Talaj és trágyvizsgálati módszerek. 1970; (48): 83–161.\n\nBarreles-Brito E, Etchevers-Barra J, Hidalgo-Moreno C, et al.: Determinación in vitro de la emisión de CO2 en muestras de mantillo in vitro determination of CO2 emission in forest litter. Agrociencia. 2014; 48(7): 679–690. Reference Source\n\nBae SL, Kwak K, Kim S, et al.: Isolation and characterization of CO2-fixing hydrogen-oxidizing marine bacteria. J Biosci Bioeng. 2001; 91(5): 442–8. PubMed Abstract \| Publisher Full Text\n\nSingla A, Inubushi K: CO2, CH4 and N2O production potential of paddy soil after biogas byproducts application under waterlogged condition. Int J Agric Environ Biotech. 2013; 6(2): 233–239. Reference Source\n\nKurek I, Reed JS, Dyson L, et al.: Engineered CO2-Fixing Chemotrophic Microorganisms Producing Carbon-Based Products and Methods of Using the Same. Patent, US20170152533 A1. 2017. Reference Source\n\nBautista G, Mátyás B: Dataset 1 in: Unexpectedresults in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity. F1000Research. 2017. Data Source\n\nBautista G, Mátyás B: Dataset 2 in: Unexpected results in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity. F1000Research. 2017. Data Source\n\nBautista G, Mátyás B: Dataset 3 in: Unexpected results in Chernozem soil respiration while measuring the effect of a bio-fertilizer on soil microbial activity. F1000Research. 2017. Data Source" }	[ { "id": "27580", "date": "20 Nov 2017", "name": "Ankit Singla", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nBautista and Matyas observed unexpected results in Chernozem soil respiration following the different fertilizer treatments. I think, the values of Dataset 2 could be directly included in the main content of the paper, if possible. The title of Dataset 1 should be \"Average values for various properties of Chernozem soil\".\n\nI have answered ‘partly’ to the question ‘Are all the source data underlying the results available to ensure full reproducibility?’ as soil ecosystems are very diverse and results could vary under different environmental conditions.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "27583", "date": "29 Nov 2017", "name": "Muhammad Aslam Ali", "expertise": [ "Reviewer Expertise Soil GHGs flux measurement", "soil microbes & environment" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n1. Why did the authors select Phylazonit biofertilizer? Does it contain any methanotrophs bacterial spp. or any electron acceptors? Didn’t find the composition. 2. Why not investigate the CO2 production rate with varying levels such as 0.5%, 1% and 5% substrates application in soils? 2. What were the initial content of organic carbon, total nitrogen, soil pH, redox status (Soil Eh) and microbial composition of the collected 24 soil samples? 3. How did the researchers control the pressure within the glass bottles during the experimental period? 4. How did the authors maintain moisture levels or water filled pore space uniformity in the 24 soil samples containing glass bottles? 5. Why didn’t you collect the gas samples evolved from the soils in glass bottles at varying time hours? 6. Why didn’t you follow the light/dark conditions in the Incubator where the glass bottles were kept? 7. All the Figures are not clear, no contrasting colors or bullets with lines used to differentiate the treatments. 8. How were soil microbial activities assessed? Methanogens and methanotroph’s relative intensity were not found in this script, which are the major focus related to the current research topic.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3218", "date": "18 Dec 2017", "name": "Gabriela Bautista", "role": "Author Response", "response": "Dear Prof. Muhammad Aslam Ali We are trying to answer your questions, and submit a second version of the manuscript in order to clarify the following points. 1. Why did the authors select Phylazonit biofertilizer? Does it contain any methanotrophs bacterial spp. or any electron acceptors? Didn’t find the composition. The Phylazonit Ltd. provided the biofertilzer for test. The Methods chapter begins with the information related to the composition: \"Bacillus megaterium, Bacillus circulans, Pseudomonas putida, in an optimized ratio for soil injection\". We did not say that the fertilizer contains methanotrophs bacterial spp. or any electron acceptors. Thats why the results presented in the paper are unexpected. 2. Why not investigate the CO2 production rate with varying levels such as 0.5%, 1% and 5% substrates application in soils? This Research note discusses only unexpected results come from an experiment that was carried out using Oxitop devices. This is part of a project in which more methods are applied. In an other method (using liquid-alkaline absorption) is possible to setup the different levels, but that is not part of the discussion of the present paper. Using Oxitop bottles only one level is possible for the setup. 2. What were the initial content of organic carbon, total nitrogen, soil pH, redox status (Soil Eh) and microbial composition of the collected 24 soil samples? In the Dataset 1: Average values for a number of different soil properties you can find the main phyical, chemical and microbial soil properties such as pH (H2O), pH (KCl), Organic carbon. We will extend the dataset with the Total Nitrogen in the second version of the paper. 3. How did the researchers control the pressure within the glass bottles during the experimental period? The Oxitop automatically measures the changes in the bottles due to gas consumption by its sensor, there is no needed to apply external measurement. 4. How did the authors maintain moisture levels or water filled pore space uniformity in the 24 soil samples containing glass bottles? The measurement was carried out in closed system (bootles), it is not possible to open the bottles during the measurement. 5. Why didn’t you collect the gas samples evolved from the soils in glass bottles at varying time hours? The Oxitop continously measures the changes. As Fig.1 and Fig.2 show during 168 hours the gas oxygen consumption/ CO2 production were measured. 6. Why didn’t you follow the light/dark conditions in the Incubator where the glass bottles were kept? In order to avoid the effect of the photosynthesis by algeas. We were interested in soil bacteria activities only. 7. All the Figures are not clear, no contrasting colors or bullets with lines used to differentiate the treatments. We do not understand this question. In Both figures we used different colors and bullets. Control (absolute): Blue Control + glucose: Red Treated: Green Treated + glucose: Purple 8. How were soil microbial activities assessed? Methanogens and methanotroph’s relative intensity were not found in this script, which are the major focus related to the current research topic. This paper was submitted as a Research note. Research notes are often preliminary studies, descriptions of unexpected and perhaps unexplained observations or lab protocols. We concluded that \"Further genomics research could detect the bacterial strains that consumed the CO2 in this soil.\" Gabriela Bautista, Bence Mátyás" } ] } ]	1	https://f1000research.com/articles/6-1950
https://f1000research.com/articles/6-1286/v1	31 Jul 17	{ "type": "Case Report", "title": "Case Report: Laparoscopic hepatectomy in an elderly patient with major comorbidities", "authors": [ "Georgios C. Sotiropoulos", "Nikolaos Machairas", "Ioannis D. Kostakis", "Georgios C. Sotiropoulos", "Nikolaos Machairas" ], "abstract": "Surgeons have been hesitant to proceed to hepatectomy in elderly patients, due to the higher rate of comorbidities and the reduced reserves. An 81-year-old male with hepatocellular carcinoma in the segment VI of the liver and several major cardiovascular, pulmonary and metabolic comorbid illnesses was referred to our department for treatment. He underwent transarterial chemoembolization of the liver tumor and afterwards he underwent laparoscopic resection of the hepatic segment VI, with an uneventful postoperative course. This case indicates the safety and feasibility of laparoscopic liver resections in older patients, even when major comorbidities are present, provided that there is a careful planning of therapeutic strategy and operation.", "keywords": [ "hepatectomy", "liver resection", "laparoscopic", "elderly", "geriatric", "comorbidity" ], "content": "Introduction\n\nLiver resection is the treatment of choice for many liver tumors. However, liver resections, and especially major hepatectomies, have been associated with several complications and the presence of comorbid illnesses has been related to increased postoperative morbidity and mortality1. Surgeons have been hesitant to proceed to hepatectomy in elderly patients, due to the higher rate of comorbidities and the reduced hepatic, cardiac, pulmonary and renal reserve, which render them more susceptible to complications. However, there are several attempts towards the adaptation of liver resections for elderly patients that have been reported in the literature, with good outcomes2. We report the case of an 81-year-old male with hepatocellular carcinoma and several comorbid illnesses who underwent laparoscopic liver segmentectomy in our department.\n\n\nCase report\n\nAn 81-year-old male patient with deteriorating right subcostal pain and high values of serum gamma-glutamyl transpeptidase and alkaline phosphatase underwent an abdominal ultrasound scan, which revealed the presence of a heterogeneous tumor 10cm in diameter, located in the right hepatic lobe, along with mild steatosis of the liver. The patient underwent an abdominal and thoracic computed tomography and an abdominal magnetic resonance imaging. They showed a heterogeneous tumor in the hepatic segment VI, which presented intense arterial uptake of the intravenous contrast followed by quick venous washout, without any sites suspicious for metastases (Figure 1). Additionally, he underwent an ultrasound-guided biopsy of the mass, which revealed the presence of a moderately differentiated hepatocellular carcinoma (HCC). Serum levels of alpha-fetoprotein (AFP) were within normal range. The patient’s medical history included arterial hypertension, type 2 diabetes mellitus, atrial fibrillation and chronic obstructive pulmonary disease, rendering him a patient with an ASA (American Society of Anesthesiologists) score 3, but no viral hepatitis, cirrhosis or any other liver pathology, apart from mild liver steatosis. The patient’s body mass index (BMI) was 26.\n\nThe patient was referred to our department for treatment. Blood gas analysis revealed the following parameters: pO2: 58mmHg, pCO2: 45mmHg, HCO3: 26mEq/L, pH: 7.38. The patient received both pulmonary and anesthesiological consultation, and he was treated with daily bronchodilations and respiratory physiotherapy. The patient initially underwent a transarterial chemoembolization of the HCC as a bridging treatment to operation. He was followed-up in an outpatient basis for about a month. His blood gas analysis showed a notable improvement: pO2: 75mmHg, pCO2: 39mmHg, HCO3: 24mEq/L, pH: 7.41. The patient was admitted to our hospital and he underwent laparoscopic resection of the hepatic segment VI, which contained the tumor, along with laparoscopic cholecystectomy. The postoperative course was uneventful and the patient was discharged on the 4th postoperative day.\n\nThe histopathological examination of the surgical specimen showed that the tumor corresponded to a moderately differentiated hepatocellular carcinoma, grade II and III in the Edmondson-Steiner grading scale, with infiltration, but not disruption of Glisson’s capsule, and without infiltration of blood vessels (pT1 tumor) (Figure 2). The resection margins were tumor-free. The histopathological examination also confirmed the mild liver steatosis that the abdominal ultrasound had indicated. The patient remains in good general condition without evidence of tumor recurrence 30 months after the operation.\n\n\nDiscussion\n\nElderly patients frequently have a fragile health, as a result of many kinds of comorbidities that present at their age that are associated with reduced reserves. The arising higher susceptibility of elderly patients to complications makes surgeons more reluctant to proceed to major operations in these patients3. Therefore, liver resections for old patients, and especially major hepatectomies, have been adopted with delay2.\n\nHowever, several studies have addressed feasibility, efficacy and safety of liver resections in elderly patients. Although there are various cut-off points for the definition of elderly patients among these studies, most of them use 704 or 75 years5–7 of age as a threshold to define older patients. All types of liver resections have been reported for patients with advanced age, from wedge resections and segmentectomies up to hemihepatectomies. Many studies have reported that there is no actual advantage regarding morbidity and mortality of younger over older patients undergoing liver resection, if older patients are considered fit enough to undergo the procedure7. Nevertheless, there are also several studies reporting an increased rate of postoperative morbidity and/or mortality for older patients than younger ones4–6.\n\nApart from the age as an independent predictor of postoperative outcomes, the existence of comorbidities has been evaluated as an important factor of worse postoperative results. Several studies have shown that patients undergoing liver resection who suffer from arterial hypertension, diabetes mellitus, arrhythmias, coronary disease, heart failure, chronic obstructive pulmonary disease, renal disease, liver cirrhosis, stroke and/or other major comorbidities have increased postoperative morbidity and/or mortality in comparison with the patients with only minor or without any comorbid diseases. An ASA score of 3 or greater has been associated with higher rates of postoperative complications and worse outcomes in general4,5.\n\nThere are only a few series reporting laparoscopic liver resections in elderly patients. When older patients (older than 70 or 75 years of age) with liver pathology undergoing laparoscopic liver resection were compared to younger ones, no significant differences were detected with regards to postoperative morbidity and mortality8,9. Furthermore, when laparoscopic and open hepatectomies were compared in elderly patients, there was no agreement concerning postoperative complications, with some authors reporting decreased rates in the case of laparoscopic hepatectomies9, whereas other authors suggested that there is no actual difference between the two approaches10. However, it is accepted that laparoscopic procedures have the advantage of less blood loss and shorter hospital stay in elderly patients9,10.\n\nOur patient aged 81 years and had some major comorbidities. However, the careful therapeutic planning with the optimization of his pulmonary status and the careful selection of the exact type of liver resection rendered the patient able to undergo the laparoscopic segmentecotmy with an uneventful postoperative course. In conclusion, laparoscopic liver resections are safe and feasible in older patients, even when major comorbidities are present, provided that there is a careful therapeutic planning.\n\n\nConsent\n\nWritten informed consent for publication of clinical details and/or clinical images was obtained from the patient.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nRussell MC: Complications following hepatectomy. Surg Oncol Clin N Am. 2015; 24(1): 73–96. PubMed Abstract \| Publisher Full Text\n\nPhan K, An VV, Ha H, et al.: Hepatic resection for malignant liver tumours in the elderly: a systematic review and meta-analysis. ANZ J Surg. 2015; 85(11): 815–22. PubMed Abstract \| Publisher Full Text\n\nKnittel JG, Wildes TS: Preoperative assessment of geriatric patients. Anesthesiol Clin. 2016; 34(1): 171–83. PubMed Abstract \| Publisher Full Text\n\nSchiergens TS, Stielow C, Schreiber S, et al.: Liver resection in the elderly: significance of comorbidities and blood loss. J Gastrointest Surg. 2014; 18(6): 1161–70. PubMed Abstract \| Publisher Full Text\n\nTzeng CW, Cooper AB, Vauthey JN, et al.: Predictors of morbidity and mortality after hepatectomy in elderly patients: analysis of 7621 NSQIP patients. HPB (Oxford). 2014; 16(5): 459–68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrcutt ST, Artinyan A, Li LT, et al.: Postoperative mortality and need for transitional care following liver resection for metastatic disease in elderly patients: a population-level analysis of 4026 patients. HPB (Oxford). 2012; 14(12): 863–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMotoyama H, Kobayashi A, Yokoyama T, et al.: Impact of advanced age on the short- and long-term outcomes in patients undergoing hepatectomy for hepatocellular carcinoma: a single-center analysis over a 20-year period. Am J Surg. 2015; 209(4): 733–41. PubMed Abstract \| Publisher Full Text\n\nNomi T, Fuks D, Kawaguchi Y, et al.: Laparoscopic major hepatectomy for colorectal liver metastases in elderly patients: a single-center, case-matched study. Surg Endosc. 2015; 29(6): 1368–75. PubMed Abstract \| Publisher Full Text\n\nCauchy F, Fuks D, Nomi T, et al.: Benefits of Laparoscopy in Elderly Patients Requiring Major Liver Resection. J Am Coll Surg. 2016; 222(2): 174–84.e10. PubMed Abstract \| Publisher Full Text\n\nWang XT, Wang HG, Duan WD, et al.: Pure Laparoscopic Versus Open Liver Resection for Primary Liver Carcinoma in Elderly Patients: A Single-Center, Case-Matched Study. Medicine (Baltimore). 2015; 94(43): e1854. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "27703", "date": "20 Nov 2017", "name": "Jun Li", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe case report by Sotiropoulos et al did show that combining the minimal invasive approach and preoperative optimal management of the comorbidities could make liver resection in very elderly patient safe. The preoperative TACE not only served as a back-up treatment for the large HCC, but also might reduce the risk of metastasis.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [ { "c_id": "3271", "date": "15 Dec 2017", "name": "Ioannis Kostakis", "role": "Author Response", "response": "We would like to thank the reviewer for his kind comments." } ] }, { "id": "28236", "date": "30 Nov 2017", "name": "Troy S. Wildes", "expertise": [ "Reviewer Expertise Preoperative care", "post-operative delirium", "anesthesia in aged patients" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide an interesting case report of an aged individual with significant comorbidities undergoing an intermediate risk surgical procedure.\nThe operative and optimization pathway are discussed in appropriate detail. The authors are also to be commended for including the patient's health status 30 months after the surgery, as such information is clearly relevant to surgical decision-making in patients with significant comorbidities.\nThe report would benefit from the following additional details and suggested edits:\nThe case presented does not justify the statement \"In conclusion, laparoscopic liver resections are safe and feasible in older patients, even when major comorbidities are present, provided that there is a careful therapeutic planning.\" Describing a medical practice as safe implies a very low event rate, which cannot be demonstrated with a single successful patient outcome.\n\nThe patient's medical comorbidities are listed. However, important functional details such as the patient's functional independence, metabolic functional capacity, and COPD symptom burden are not described. Such details would be critically informative of the comorbidity disease burden, frailty status, and anticipated complication rate of the patient. Additionally, inclusion of other key diagnostic information, such as serum creatinine and hemoglobin, would be relevant along with the above information for predicting pulmonary and acute kidney injury risk. Without inclusion of all of this information, it is difficult for the reader to truly appreciate the risk level of this patient.\n\nIs the background of the case’s history and progression described in sufficient detail? Partly\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? No\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Partly\n\nIs the case presented with sufficient detail to be useful for other practitioners? Partly", "responses": [ { "c_id": "3270", "date": "18 Dec 2017", "name": "Ioannis Kostakis", "role": "Author Response", "response": "Comment 1The case presented does not justify the statement \"In conclusion, laparoscopic liver resections are safe and feasible in older patients, even when major comorbidities are present, provided that there is a careful therapeutic planning.\" Describing a medical practice as safe implies a very low event rate, which cannot be demonstrated with a single successful patient outcome. Answer We removed this sentence and we replaced it with the following sentence: “Laparoscopic liver resections could be applied even to elderly patients with major comorbidities after optimization of their medical status”. Comment 2The patient's medical comorbidities are listed. However, important functional details such as the patient's functional independence, metabolic functional capacity, and COPD symptom burden are not described. Such details would be critically informative of the comorbidity disease burden, frailty status, and anticipated complication rate of the patient. Additionally, inclusion of other key diagnostic information, such as serum creatinine and hemoglobin, would be relevant along with the above information for predicting pulmonary and acute kidney injury risk. Without inclusion of all of this information, it is difficult for the reader to truly appreciate the risk level of this patient. Answer We added data regarding patient’s functional independence, ability for physical activity, creatinine and urea serum levels, hematocrit, COPD symptoms and results of spirometry." } ] } ]	1	https://f1000research.com/articles/6-1286
https://f1000research.com/articles/6-1750/v1	25 Sep 17	{ "type": "Data Note", "title": "First de novo draft genome sequence of Oryza coarctata, the only halophytic species in the genus Oryza", "authors": [ "Tapan Kumar Mondal", "Hukam Chand Rawal", "Kishor Gaikwad", "Tilak Raj Sharma", "Nagendra Kumar Singh", "Hukam Chand Rawal", "Kishor Gaikwad", "Tilak Raj Sharma", "Nagendra Kumar Singh" ], "abstract": "Oryza coarctata plants, collected from Sundarban delta of West Bengal, India, have been used in the present study to generate draft genome sequences, employing the hybrid genome assembly with Illumina reads and third generation Oxford Nanopore sequencing technology. We report for the first time that more than 85.71 % of the genome coverage and the data have been deposited in NCBI SRA, with BioProject ID PRJNA396417.", "keywords": [ "Abiotic stress", "Genome assembly", "Halophyte", "Nanopore", "NGS", "Salt stress", "Wild Oryza", "Whole genome sequencing" ], "content": "Introduction\n\nSoil salinity is a major abiotic stress of rice cultivation globally (Molla et al., 2015), and rice cultivation areas under soil salinity stress are increasing gradually. Genetic potential for salt tolerance of rice that exists among the natural population has been largely exploited, and alternative useful alleles may further enhance salinity tolerance. Wild species are a potential source of many useful genes and QTLs that may not be present in the gene pool of the domesticated species.\n\nOryza coarctata, known as Asian wild rice, grows naturally in the coastal region of South-East Asian countries. It flowers and set seeds under as high as 40 E.Ce dS m-1 saline soil (Bal & Dutt, 1986). It is the only species in the genus Oryza that is halophyte in nature. However, with the exception of one transcriptomic (Garg et al., 2014) and one miRNA (Mondal et al., 2014) experiment, no large scale generation of any other genomic resource is available for this important species, although several pinitol biosynthesis pathway genes have been cloned to study the functional genomics (Sengupta & Majumder, 2009).\n\n\nMethods\n\nThe plants were collected from its native place, Sundarban delta of West Bengal, India (21º.36'N and 88º.15' E) and established to our institute NET house. To determine the genome size, 20 mg of young leaf tissue from Net house grown plants was chopped into small pieces and stained with RNase containing propidium iodide (50 μg/ml) (BD Science, India) as per the protocol of Dolezel et al. (2007). The samples were filtered through a 40-μM mesh sieve (Corning, USA), before analysis in (CFM) BD FACS Calibur (BD Biosciences, San Jose, CA, USA). Pisium sativum leaf was used as standard for calculating the genome size. Further, high-quality genomic DNA from 100 mg young leaf was extracted using CTAB method (Ganie et al., 2016) for the preparation of various genomic DNA libraries. We used Illumina 4000 GA IIx sequencer (San Diego, CA, USA), with 150-bp paired-end libraries, four mate-pair library (with 150-bp paired-end libraries) of four different sizes (average of 2, 4, 6 and 10 kb size). In addition, we also used third generation sequencing (Oxford Nanopore) technology for better assembly. Sequencing was performed on MinION Mk1b (Oxford Nanopore Technologies, Oxford, UK) using SpotON flow cell (R9.4) in a 48h sequencing protocol on MinKNOW 1.4.32. Base calling was performed using Albacore. Base called reads were processed using poRe version 0.24 (Watson et al., 2015) and poretools version 0.6.0 (Loman & Quinlan, 2014). The simple sequence repeats (SSRs) of each scaffold were identified by MISA perl script (Thiel et al., 2003). Gene model prediction was done by AUGUSTUS 3.1 (Stanke & Waak, 2003) and genes were functionally analysed using InterProScan version 5.16.55 (Jones et al., 2014). The InterProScan results were further parsed for additional functional evidence (GO terms and KEGG pathway) using interproscanParser script available at iPlant (Brozynska et al., 2016). Noncoding RNAs, such as miRNA, tRNA, rRNA, snoRNA, snRNA, were identified by adopting Infernal v1.1.2 (Nawrocki & Eddy, 2013) using Rfam (Nawrocki et al., 2015). Transfer RNA was predicted using tRNAscane-SE v 1.23 (Schattner et al., 2005)\n\n\nDiscussion\n\nThe genome (KKLL) of O. coarctata is tetraploid (2n=4X=48) with a genome size estimated by flow cytometer is found to be approximately 665Mb. The Illumina 4000 GA IIx sequencer pair-end generated 137 Gb data. Further four mate-pair libraries together generated 104.35 Gb and Nanopore generated 6.35 Gb sequence data. Hence, we achieved 372.48 X depth of the genome of O. coarctata. The final assembly generated 58362 number of contigs with a minimum length of 200 bp to maximum length of 7,855,609 bp and 1,858,627 bp N50 value, making a total contig length of 569994164 (around 570 Mb) assembled genome, resulting 85.71 % genome coverage. It has been calculated that data contain very small amount of non-ATGC character. Further, we also found that the repeat contain 19.89% of the genome. We also identified approximately 1605 different non-coding RNAs and around 105673 SSRs. Gene ontology analysis identified several salt responsive genes.\n\n\nData availability\n\nRaw sequence data are available at NCBI SRA under the BioProject ID: PRJNA396417.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nTKM is also grateful to Mr Sukdev Nath, who provided the planting material. TRS is thankful to the DST, Govt. of India for JC Bose National Fellowship. The authors are thankful to M/S Genotypic Technology Private Limited, Bengaluru, India for sequencing work and M/S BD Biosciences, India for Flow Cytometer work.\n\n\nReferences\n\nBal AR, Dutt SK: Mechanism of salt tolerance in wild rice (Oryza coarctata Roxb). Plant Soil. 1986; 92(3): 399–404. Publisher Full Text\n\nBrozynska M, Copetti D, Furtado A, et al.: Sequencing of Australian wild rice genomes reveals ancestral relationships with domesticated rice. Plant Biotechnol J. 2017; 15(6): 765–774. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDolezel J, Greilhuber J, Suda J: Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007; 2(9): 2233–2244. PubMed Abstract \| Publisher Full Text\n\nGanie SA, Borgohain MJ, Kritika K, et al.: Assessment of genetic diversity of Saltol QTL among the rice (Oryza sativa L.) genotypes. Physiol Mol Biol Plants. 2016; 22(1): 107–114. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarg R, Verma M, Agrawal S, et al.: Deep transcriptome sequencing of wild halophyte rice, Porteresia coarctata, provides novel insights into the salinity and submergence tolerance factors. DNA Res. 2014; 21(1): 69–84. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLoman NJ, Quinlan AR: Poretools: a toolkit for analyzing nanopore sequence data. Bioinformatics. 2014; 30(23): 3399–3401. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMolla KA, Debnath AB, Ganie SA, et al.: Identification and analysis of novel salt responsive candidate gene based SSRs (cgSSRs) from rice (Oryza sativa L.). BMC Plant Biol. 2015; 15: 122. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMondal TK, Ganie SA, Debnath AB: Identification of novel and conserved miRNAs from extreme halophyte, Oryza coarctata, a wild relative of rice. PLoS One. 2015; 10(10): e0140675. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNawrocki EP, Eddy SR: Infernal 1.1: 100-fold faster RNA homology searches. Bioinformatics. 2013; 29(22): 2933–2935. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNawrocki EP, Burge SW, Bateman A, et al.: Rfam 12.0: updates to the RNA families database. Nucleic Acids Res. 2015; 43(Database issue): D130–7. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSengupta S, Majumder AL: Insight into the salt tolerance factors of a wild halophytic rice, Porteresia coarctata: a physiological and proteomic approach. Planta. 2009; 229(4): 911–929. PubMed Abstract \| Publisher Full Text\n\nStanke M, Waack S: Gene prediction with a hidden Markov model and a new intron submodel. Bioinformatics. 2003; 19(Suppl 2): ii215–225. PubMed Abstract \| Publisher Full Text\n\nThiel T, Michalek W, Varshney RK, et al.: Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet. 2003; 106(3): 411–422. PubMed Abstract \| Publisher Full Text\n\nWatson M, Thomson M, Risse J, et al.: poRe: an R package for the visualization and analysis of nanopore sequencing data. Bioinformatics. 2015; 31(1): 114–115. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "26360", "date": "11 Oct 2017", "name": "Sandip Das", "expertise": [ "Reviewer Expertise Comparative genomics", "brassica", "polyploidy", "regulatory evolution" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a draft genome sequence of a halophyte Oryza species collected from Sunderbans, and provides a glimpse into the adaptive strategies employed by Oryza against salinity stress. Undoubtedly, it will be an useful resource for future functional characterization, comparative genomic studies, and developing salinity tolerance in rice. I understand that the present format is only for reporting, and look forward to reading the full manuscript with all the analysis. There are small language edits that that authors need to incorporate.\n\nComments: Please add a reference or sufficient information for general readers as to how genome types of rice (for instance, KKLL in case of O. coarctata) was assigned.\nLanguage corrections:\nChange “have been used” to “has been used” Change “We report for the first time that more than 85.71 % of the genome coverage and the data have been deposited in NCBI SRA, with BioProject ID PRJNA396417” to “deposited in NCBI SRA, with BioProject ID PRJNA396417” Change “and established to our institute NET” to “and established at our institute NET” Change “resulting 85.71 % genome coverage” to “resulting in 85.71 % genome coverage” Change “we also found that the repeat contain 19.89% of the genome.” to “we also found that the 19.89% of the genome is repetitive in nature”.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "26486", "date": "13 Oct 2017", "name": "Stephen P. Moose", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors report a whole genome sequence dataset for a halophytic wild rice species. These data will be useful for discovery of novel alleles for rice improvement, and for comparative/evolutionary genomics within the Oryza genus.\n\nThe report would benefit from more details on the plant accession used as source of DNA for sequencing. It is stated O. coarctata is tetraploid. Was that determined by the authors, or is there a citation to include? Is it known whether O. coarctata is typically self or cross-pollinated, or other information about expected degree of heterozygosity? When grown in greenhouse to generate the plant tissue used for DNA extraction, were the plant(s) established from seeds, or via clonal propagation? Was the genomic DNA used to prepare sequencing libraries from a single plant, or a pool from multiple plants? This information is important to assess expected frequencies of variant types such as alleles or homeologs due to tetraploidy, which are likely collapsed to varying degrees in the subsequent assembly.\n\nThere is mention of an assembly and its quality, but not about the method(s) used to produce it or key parameters that guided the assembly. Can the authors provide that information, so that others have a benchmark upon which to compare future assemblies using the datasets?\n\nThe sentence “Further, we also found that the repeat contain 19.89% of the genome.” Is not completely clear. I believe what the authors intend to say is that approximately 20% of the genome assembly is comprised of repeats. How was this sequence fraction defined as repeats, via tool for matching to known repeat sequences, or a de novo approach? By inference, it is also likely that the approximately 100-kb of the estimated genome size not covered by the assembly is comprised of high-copy repeats, leading to an estimate of about 30% total repeat content.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] }, { "id": "26359", "date": "20 Oct 2017", "name": "Kashmir Singh", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe work describe the whole genome sequence of wild species of Oryza coarctata species that exclusively grow under saline water and thus will be an important source of salinity tolerance genes. These genes can later be used to introduce salinity tolerance in commercial cultivars of rice. The authors used Illumina and Oxford nanopore sequencing platforms to generate 372.48X data.\nThe genome sequencing methods seems good enough but authors have discussed very little about the annotation of the genome data. I can understand that there is word limit under Data Note in F1000Research, but still by looking at the discussion, I think analysis portion is weak point in this paper. Authors should provide a comparative note on the genome of Oryza sativa and Oryza coarctata. How this species is tolerating such a high saline conditions, which kind of genes/osmoregulators are involved in this adaptation should be discussed along with comparison to O. sativa. How many different genes were predicted should be mentioned. Authors found approximately 1605 non-coding RNAs? I am not sure, what are trying to tell here, this number should be high as per my opinion.\nThere are some minor mistakes like; in the affiliation the word “Delhi” is not required. The word, “Primary” should be inserted in the first paragraph last line of Introduction. So the correct sentence will be \"...in the primary gene pool...\".\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-1750
https://f1000research.com/articles/6-2129/v1	13 Dec 17	{ "type": "Correspondence", "title": "Can circulating tumor DNA be used for direct and early stage cancer detection?", "authors": [ "Eleftherios P. Diamandis", "Clare Fiala", "Clare Fiala" ], "abstract": "In the August 16th issue of Science Translational Medicine, Phallen et al propose a method for early cancer diagnosis by using circulating tumor DNA (1). One major advance of this paper includes optimized sequencing of cell-free/circulating tumor DNA (ctDNA) without knowledge of tumor mutations. Evaluation of 200 patients with colorectal, breast, lung and ovarian cancer revealed mutations in ctDNA in approx. 60-70% of all patients, including stage 1 and stage 2 disease. If this data can be reproduced in asymptomatic individuals, they will likely have a major impact on early cancer detection and patient outcomes. In this commentary, we examine the feasibility of this approach for detecting small, asymptomatic tumors, based on previously published empirical data.", "keywords": [ "Circulating tumor", "Cancer diagnosis", "Cancer detection", "Tumor measurement", "Colorectal", "breast", "lung", "ovarian cancer data" ], "content": "Correspondence\n\nPhallen et al. recently proposed a method for early cancer diagnosis by using circulating tumor DNA1. One major advance of this paper includes optimized sequencing of cell-free/circulating tumor DNA (ctDNA) without knowledge of tumor mutations. Evaluation of 200 patients with colorectal, breast, lung and ovarian cancer revealed mutations in ctDNA in approximately 60–70% of all patients, including stage 1 and stage 2 disease. If this data can be reproduced in asymptomatic individuals, they will likely have a major impact on early cancer detection and patient outcomes. In this correspondence, we examine if this approach is effective in detecting small, asymptomatic tumors, based on previously published empirical data.\n\nAn important question is the desirable size of a tumor to be detected with such methods. For this discussion, we will use data mostly from breast cancer, for which there is ample experience from screening programs. Some organizations set a tumor diameter of 1 mm as the goal of early detection2 since tumors of this size are localized, less complex and likely to be cured by radical resection. The literature suggests that the chances of progression of these small, 1 mm diameter tumors, is less than 1%3. However, such small tumors could be found in many asymptomatic individuals and this may lead to over-diagnosis and over-treatment3. Consequently, we also include larger tumors in this discussion. It could be argued that 5 mm in diameter is an optimal tumor size for early and curable cancer detection. At 5 mm diameter, the chances of this tumor progressing are small (around 6%)3, and currently, these small lesions are only detectable by mammography (or other imaging modalities) in only 26% of cases4. We also suggest that detection of a 10 mm diameter tumor may not be an advance in breast cancer screening, since at this size, the chances of progression increase considerably (to about 50%), and most of these tumors (> 90%), are currently detected by mammographic screening.\n\nWe further assume that the circulating free DNA concentration (cfDNA) in individuals without cancer, or very small cancers (ctDNA), is about 5 ng/mL on average5, which is equivalent to about 6,000 whole haploid genomes per 4 mL of plasma (10 mL blood draw). For this discussion we also use well-established measurements of tumor volume and cellularity. A tumor of approximately 12.5 mm in diameter, weights approx. 1 gram, has a volume of 1 cm3 and contains approx. 100 million to 1 billion cells6. According to recent estimates of ctDNA and tumor volume, a 10 gram tumor has a volume of 10 cm37. For such tumors, the average percent fraction of mutant DNA has been reported to be 0.1 %, or 1 mutant DNA molecule per 1,000 non-mutated DNA molecules in the circulation7.\n\nBased on these published assumptions, we constructed Table 1.\n\n1. As reported in Ref 7; bold font indicates experimental data. Other data were calculated by extrapolation\n\n2. As reported in Ref 6\n\n3. As reported in Ref 3\n\n4. As reported in Ref. 4\n\nTable 1 shows the minimum tumor measurements required for 4 mL of plasma to contain at least 1 mutated genome. The percent fraction of mutant DNA is approximately 1 in 10,000 (0.01%) which corresponds to a tumor volume of 1 cm3 or 12.5 mm in diameter (assuming a spherical nodule). Most, if not all, of these tumors are currently detectable by mammographic screening or other imaging modalities, which have an approximate limit of detection of about 4 mm diameter8. Table 1 also shows that when the tumor diameter drops below 10 mm, the chances of this method working are minimal, since there will be not enough tumor DNA (at least one copy) in 4 mL of plasma to make detection possible. Consequently, Table 1 predicts that 5 mm diameter tumors will not be detected due to this sampling error. These estimates are corroborated by the data presented by Phallen et al.1. They used tumors of varying sizes, stages and types, but universally, the mutant fraction of ctDNA is never less than 0.01%, the approximate threshold for 1 genome copy in 4 mL of plasma. We do not know if the patients included in the study have been preselected to have a specific cut-off of percent mutant DNA in the circulation. If this is the case, the data will be biased towards detection of tumors which are rather large (> 10 mm in diameter and have a mutant fraction of > 0.01%). In fact, >95% of their cases have mutant fraction > 0.1% (Figure 5 from Phallen et al.1 which suggests tumor sizes of > 27 mm (Table 1). We anticipate that in a real case scenario, small and asymptomatic tumors will likely yield percent mutant DNA fraction of much less than 0.1%, thus making detection of these tumors highly unlikely, due to sampling error.\n\nThe sensitivity of the method proposed by Phallen et al. is further compromised by the number of genes tested for mutations (currently 58 genes). The predicted maximum sensitivity is about 80%, assuming best case scenario of 1 mutation detected to signify cancer. If we assume that the overall positivity of this test is 50%, then, the overall sensitivity will likely be less than 40%. This would make screening of asymptomatic individuals less efficient, since the majority of patients will receive incorrect screening results (false negatives) due to either lack of the targeted mutations or sampling error.\n\nThe specificity of this method is claimed to be very high due to detection of only driver mutations. However, as shown in Phallen et al.’s Supplementary Figure S31, the gene with the highest sensitivity for cancer detection of all of the 4 cancer types is p53. However, there are multiple reports demonstrating mutations of p53 in apparently healthy individuals, ranging from 0.2% to 11%8–11. If we assume an overall specificity of 99% and an overall sensitivity of 40% with this test, and a prevalence of cancer around 1%, then in 1,000 screened individuals, there will be 4 true positive, 6 false negative, 10 false positive and 992 true negative results. The positive predictive value (PPV) will be only 29%. At 98% specificity, the PPV further drops to 20%. Not only would more than half the patients will receive incorrect (false negative) results but about 72–80% of tested positive patients would need to undergo additional diagnostic procedures to rule-in or rule-out cancer. Additional specificity concerns may include technical noise and somatic mutations stemming from other biological phenomena. Also, in this paper, no matched DNA was employed to differentiate somatic from germline mutations, another important limitation12.\n\nOur analysis shows that measurement of ctDNA for early cancer diagnosis is problematic, not only due to the limited capability of deep whole genome sequencing to reveal mutations, but because the amount of circulating tumor DNA retrieved from a 10mL blood draw will be extremely small, or even nonexistent, thus making efficient diagnosis of cancer unlikely. Our analyses generally show that when the tumor diameter drops below 10 mm, not a single mutant DNA copy will be retrieved. We do not exclude the possibility of future additional technical improvements, so that mutant DNA could somehow be extracted from the whole circulation and subjected to such deep sequencing, to increase sensitivity, provided that the specificity of the test would be preserved to close to 100%. Otherwise, under a screening scenario, these methods are unlikely to work. Our analyses also show that a 1 mm tumor is likely associated with 1 copy of ctDNA in the entire circulation, further underlining the difficulty in detection. The possibility exists that early cancer detection may be possible in specific clinical contexts or specific tumor types using this method. Also, it may be possible to combine protein and molecular markers to increase sensitivity, as shown already for pancreatic cancer13.\n\nWe hope that our analyses will help to further understand the opportunities and limitations of ctDNA as a cancer biomarker, especially for early cancer detection.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was funded by a grant to EPD from the Canadian Cancer Society (Grant #703873).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nPhallen J, Sausen M, Adleff V, et al.: Direct detection of early-stage cancers using circulating tumor DNA. Sci Transl Med. 2017; 9(403): pii: eaan2415. PubMed Abstract \| Publisher Full Text\n\nGarber K: Ontario institute offers new model of cancer research. J Natl Cancer Inst. 2008; 100(14): 980–982. PubMed Abstract \| Publisher Full Text\n\nCountercurrents Series, Narod SA: Disappearing breast cancers. Curr Oncol. 2012; 19(2): 59–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWeedon-Fekjaer H, Lindqvist BH, Vatten LJ, et al.: Breast cancer tumor growth estimated through mammography screening data. Breast Cancer Res. 2008; 10(3): R41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWan JCM, Massie C, Garcia-Corbacho J, et al.: Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017; 17(4): 223–238. PubMed Abstract \| Publisher Full Text\n\nDel Monte U: Does the cell number 109 still really fit one gram of tumor tissue? Cell Cycle. 2009; 8(3): 505–506. PubMed Abstract \| Publisher Full Text\n\nAbbosh C, Birkbak NJ, Wilson GA, et al.: Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature. 2017; 545(7655): 446–451. PubMed Abstract \| Publisher Full Text\n\nSchwaederle M, Husain H, Fanta PT, et al.: Detection rate of actionable mutations in diverse cancers using a biopsy-free (blood) circulating tumor cell DNA assay. Oncotarget. 2016; 7(9): 9707–9717. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGormally E, Vineis P, Matullo G, et al.: TP53 and KRAS2 mutations in plasma DNA of healthy subjects and subsequent cancer occurrence: a prospective study. Cancer Res. 2006; 66(13): 6871–6876. PubMed Abstract \| Publisher Full Text\n\nFernandez-Cuesta L, Perdomo S, Avogbe PH, et al.: Identification of Circulating Tumor DNA for the Early Detection of Small-cell Lung Cancer. EBioMedicine. 2016; 10: 117–123. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNewman AM, Lovejoy AF, Klass DM, et al.: Integrated digital error suppression for improved detection of circulating tumor DNA. Nat Biotechnol. 2016; 34(5): 547–555. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJones S, Anagnostou V, Lytle K, et al.: Personalized genomic analyses for cancer mutation discovery and interpretation. Sci Transl Med. 2015; 7(283): 283ra53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCohen JD, Javed AA, Thoburn C, et al.: Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers. Proc Natl Acad Sci U S A. 2017; 114(38): 10202–10207. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "29018", "date": "14 Dec 2017", "name": "Nicholas J. Wald", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper is a commentary on a study by Phallen et al. who developed and applied advanced DNA analysis techniques for circulating tumor DNA from patients with untreated breast, colorectal and ovarian cancer. They were able to detect cancer in about 60-70% of patients with Stage I or Stage II disease. Diamandis and Fiala’s Commentary examines the feasibility of applying these techniques to small tumors that are not reliably detectable by other means. They extrapolate original data from Phallen et al., making the assumption that the number of tumor-derived genomes in the circulation is proportional to tumor volume. Diamandis and Fiala reasonably conclude that tumors smaller than about 10mm in diameter result in less than one tumor-derived genome per 10ml blood draw and are therefore, in principle, undetectable in practice. This logic is probably conservatively based because small, low grade tumors are likely to produce proportionally less ctDNA than high grade tumors. This Commentary is a useful contribution to the literature.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "29016", "date": "04 Jan 2018", "name": "Catherine Alix-Panabières", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis commentary, very well written by the expert Eleftherios P Diamandis and his colleague Clare Fiala, is based on the last article of Phallen et al. who described a method for early cancer diagnosis by using circulating tumor DNA (ctDNA) sequenced without knowledge of tumor mutations.\nIn this commentary, the authors examined step by step the feasibility of this approach for detecting small, asymptomatic tumors, based on previously published empirical data. They nicely showed that measurement of ctDNA for early cancer diagnosis is problematic, not only due to the limited capability of deep whole genome sequencing to reveal mutations, but because the amount of circulating tumor DNA retrieved from a 10mL blood draw will be extremely small, or even nonexistent, thus making efficient diagnosis of cancer unlikely.\nThis commentary, very well documented, is important in the liquid biopsy field and needs to be indexed to understand the current opportunities and limitations of ctDNA as a biomarker for early detection of cancer.\n\nIs the rationale for commenting on the previous publication clearly described? Yes\n\nAre any opinions stated well-argued, clear and cogent? Yes\n\nAre arguments sufficiently supported by evidence from the published literature or by new data and results? Yes\n\nIs the conclusion balanced and justified on the basis of the presented arguments? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2129
https://f1000research.com/articles/6-1968/v1	07 Nov 17	{ "type": "Research Article", "title": "Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective", "authors": [ "Ivan Nombela", "Aurora Carrion", "Sara Puente-Marin", "Veronica Chico", "Luis Mercado", "Luis Perez", "Julio Coll", "Maria del Mar Ortega-Villaizan", "Ivan Nombela", "Aurora Carrion", "Sara Puente-Marin", "Veronica Chico", "Luis Mercado", "Luis Perez", "Julio Coll" ], "abstract": "Background: Some fish viruses, such as piscine orthoreovirus and infectious salmon anemia virus, target red blood cells (RBCs), highly replicate inside them and induce an immune response. However, the implications of RBCs in the context of birnavirus infection (i.e, infectious pancreatic necrosis virus (IPNV)) have not yet been studied. Methods: Ex vivo trout RBCs were obtained from peripheral blood, ficoll purified and exposed to IPNV in order to analyze infectivity and induced immune response using RT-qPCR, immune fluorescence imaging, flow cytometry and western-blotting techniques. Results: IPNV could not infect RBCs; however, IPNV-exposed RBCs increased the expression of the INF1-related genes ifn-1, pkr and mx genes. Moreover, conditioned media from IPNV-exposed RBCs conferred protection against IPNV infection in CHSE-214 fish cell line. Conclusions: Trout RBCs could trigger an antiviral immune response against IPNV infection despite not being infected. Fish RBCs could be considered mediators of the antiviral response and therefore targets of novel DNA vaccines and new strategies against fish viral infections. Further research is ongoing to completely understand the molecular mechanism that triggers this immune response in trout RBCs.", "keywords": [ "erythrocytes", "IPNV", "birnavirus", "immune response", "antiviral", "trout", "interferon" ], "content": "Introduction\n\nViral diseases are one of the main agents that cause loses in aquaculture. Among all the viral diseases, infectious pancreatic necrosis (IPN) is a highly transmissible viral disease with a high impact in the salmon aquaculture industry. Infectious pancreatic necrosis virus (IPNV) is the responsible agent of IPN. IPNV was the first fish virus isolated in cell culture1 and has different serotypes resulting from mutations in the VP2 gene2. IPNV outbreaks are usually related to high mortality rates in salmonid aquaculture, especially in young fish3,4, highlighting the urgent necessity for the development of efficient strategies in vaccination. IPNV belongs to the Aquabirnaviridae genera within the Birnaviridae family. Virions from this family are non-enveloped with a double stranded RNA genome. This genome consists of two segments: the A segment contains the information to encode a protein that is post-translationally cleaved into VP2, VP3 and VP4 viral proteins; the B segment encodes VP1 viral protein, which is a RNA polymerase5. VP2 and VP3 are the major structural and immunogenic proteins, as they represent 64% of the total proteins of the virions6. The VP2 epitopes are the target for antibodies against different serotypes6. The VP3 role is unknown, but it could be an internal structural protein7.\n\nIn contrast to mammals, fish, reptile and avian red blood cells (RBCs) are nucleated. This implies that nucleated RBCs can support viral replication. For example, fish RBCs are the target of some fish viruses, such as infectious salmon anemia virus (ISAV)8 and piscine orthoreovirus (PRV)9,10, where viral factories and high viral titters have been observed. Moreover, both viruses can induce immune responses in infected RBCs, characterized by the expression of genes related to IFN-1 (type I interferon) pathway and IFN-γ.\n\nTypically, the role associated with RBCs is the transport of O2 to different tissues and gas exchange. The fact that these cells are nucleated implies that fish RBCs can respond to different stimulus and control cellular processes. At present, a whole set of biological processes related to the immune response has been reported in RBCs from different species: recognition of pathogen associated molecular patterns11,12 through expression of pattern recognition receptors, such as toll-like receptors (TLRs)13; production of cytokine-like factors8,12,14,15; phagocytosis16; and formation of complement immune complexes17. Recently, it has been shown that viral hemorrhagic septicemia virus (VHSV) can induce cytokine production and crosstalk with a spleen stromal cell line (unpublished report, Nombela I, Puente-Marin S, Chico V, Villena A, Carracedo B, Ciordia S, Mena M, Mercado L, Perez L, Coll J, Estepa A, Ortega-Villaizan M). Human RBCs have also been involved in the antiviral response, since it has been reported that RBCs could bind HIV-1 particles to their surface, forming complexes to ease the clearance of the viral particles18.\n\nBased on this evidence, the aim of this study was to evaluate the immune response of trout RBCs against IPNV, one of the most ubiquitous viral fish pathogens. To reach this objective, we first analyzed the infectivity of IPNV in trout RBCs. Then, the RBCs’ immune responses were evaluated in ex vivo RBCs exposed to IPNV by means of gene and protein expression methods. Finally, the ability of RBCs to protect against IPNV infection in CHSE-214 (Chinook salmon embryo) cell line, which is susceptible to IPNV infection, was evaluated using conditioned medium (CM) from IPNV-exposed RBCs. Here, we report the induction of an immune response in RBCs by IPNV, a non-infecting virus on these cells, characterized by the expression of genes related with the IFN-1 pathway, Mx production and protection against IPNV infection in CHSE-214 cells.\n\n\nMethods\n\nRainbow trout (Oncorhynchus mykiss) individuals of approximately 10 g were obtained from a commercial fish farm (PISZOLLA S.L., CIMBALLA FISH FARM, Zaragoza, Spain) with a re-circulating dechlorinated-water system, at a stocking density of 1fish/3L, and fed daily with a commercial diet (SKRETTING, Burgos, Spain) and maintained at the University Miguel Hernandez (UMH) facilities at 14°C. Fish were acclimatized to laboratory conditions over 2 weeks before experimentation.\n\nRainbow trout were sacrificed by overexposure to tricaine methanesulfonate (Sigma-Aldrich, Madrid, Spain) at 0.2 g/L. Peripheral blood was sampled from the caudal vein of rainbow trout using insulin syringes (NIPRO Bridgewater, NJ). Blood samples were placed in RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific Inc., Carlsbad, CA) supplemented with 10% FBS (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM L-glutamine (Gibco), 50 µg/mL gentamicin (Gibco), 2 µg/mL fungizone (Gibco) and 100 U/mL penicillin/streptomycin (Sigma-Aldrich). Then, RBCs were purified by two consecutive density gradient centrifugations with Histopaque 1077 (7206g, Ficoll 1.007; Sigma-Aldrich). Finally, RBCs were washed twice with RPMI 2%. Purity of RBCs of 99.9% was estimated by optical microscopy evaluation. Then, purified RBCs were cultured in the above indicated medium at a density of 107 cells/mL, in cell culture flasks, at 14°C, overnight.\n\nEx vivo rainbow trout (Oncorhynchus mykiss) RBCs along with CHSE-214 cell line (Chinook Salmon Embryo, ATCC CRL-1681) were infected using IPNV Sp strain19. IPNV was grown as previously described20. Ex vivo RBCs exposure to IPNV was performed by incubating RBCs with diluted IPNV at the indicated MOI (multiplicity of infection) in RPMI 2% FBS. After three hours of incubation, RBCs were centrifuged at 1600 rpm for 5 minutes and then washed with medium to completely eliminate the non-adsorbed excess of virus. Finally, RBCs were placed in 24 well plates (Corning Costar, Sigma-Aldrich, Madrid, Spain) with 500 µl of RPMI 2%. The whole process was done at 14ºC. Infection of the CHSE-214 cell line was done by incubating IPNV diluted in RPMI 2% at the desired MOI for 1 hour at 14ºC. After that, RPMI was retired and RPMI 2% was added to each well. Infected CHSE-214 cells were maintained at 14ºC20.\n\nIn time course experiments, the initial supernatant with IPNV was not removed. When each of the time points were reached, RBCs were washed with cell culture medium and CHSE-214 cells with PBS supplemented with calcium.\n\nThe virus titer in IPNV-exposed RBCs supernatants was determined by TCID50 quantification and RT-qPCR. Briefly, different dilutions of the supernatants (from 10-1 to 10-4) were added to CHSE-214 cell monolayers, and incubated at 14ºC for 90 minutes. Then, the virus was removed and infected CHSE-214 cell monolayers covered with a solution of RPMI 2% FBS. Cell plates were incubated at 14ºC for 7 days. For RT-qPCR titration, 30 µL of IPNV with known titer (109 TCID50/mL) and 30 µL of IPNV-exposed RBCs supernatants were used to extract RNA and synthetize cDNA, as explained hereafter. Ten-fold serial dilutions from 108 to 102 TCID50/mL were done to obtain IPNV cDNA and create a standard line.\n\nThe E.Z.N.A.® Total RNA Kit (Omega Bio-Tek Inc., Norcross, GA) was used for total RNA extraction, in accordance with manufacturer’s instructions. DNAse treatment was done in order to eliminate residual genomic DNA using TURBO™ DNase (Ambion, Thermo Fischer Scientific Inc.), following the manufacturer’s instructions. RNA was quantified with a NanoDrop® 377 Spectrophotometer (Nanodrop Technologies, Wilmington, DE).\n\ncDNA was synthetized from RNA using M-MLV reverse transcriptase (Invitrogen, Thermo Fischer Scientific Inc.), as previously described21. Final concentration of cDNA was 6 ng/µL. RT-qPCR reactions were performed in a total volume of 20 μl using 12 ng of cDNA, 10 μl of TaqMan universal PCR master mix (Thermo Fischer Scientific), 900 nM final concentration of each primer (300 nM for IPNV segment A) and 300 nM of probe (150 nM for IPNV segment A). RT-qPCR was performed using the ABI PRISM 7300 System (Thermo Fischer Scientific). Cycling conditions were 50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min.\n\nGene expression was analyzed by the 2-ΔΔCt method22. The eukaryotic 18S rRNA gene (Cat#4310893E, Thermo Fischer Scientific) was used as endogenous control. Primers and probes are listed in Table 1.\n\nFor intracellular staining, mouse polyclonal antibodies against trout IL1β (RRID: AB_2716269)23,24, IL8 (RRID: AB_2716272)25 and TNF-α (RRID: AB_2716270)26 were produced at the laboratory of Dr. Luis Mercado. Rabbit polyclonal antibody against trout Mx3 (RRID: AB_2716267)27,28 was produced at the laboratory of Dr. Amparo Estepa. Anti-IPNV-VP3 monoclonal antibody 2F12 (RRID: AB_2716296) was used for IPNV labelling29. Anti-rabbit IgG (H+L) CF™ 488 antibody produced in goat and anti-mouse IgG (H+L) CF™ 488 antibody produced in goat were used as secondary antibodies for proteins and anti-mouse IgG (H+L) CFTM 647 produced in goat to detect 2F12 antibody.\n\nFor western blotting, rabbit polyclonal antibody against human eIF2α-P (Cat# E2152, RRID:AB_259283) and rabbit polyclonal antibody against human α-Actin (Cat#2066, RRID:AB_476693) were purchased from Sigma-Aldrich.\n\nControl and IPNV-exposed RBCs pellets (≈107 cells) were used for protein extraction. Cell pellets were washed 3 times with PBS and then resuspended in 30 µl of PBS with a cocktail of protease inhibitors (Sigma-Aldrich). Then, cells were frozen/thawed 3 times and lysed using an eppendorf micropistile (Eppendorf, Hamburg, Germany). Samples were loaded in Tris–Glycine sodium dodecyl sulfate 12% polyacrylamide gels under reducing conditions. Electrophoresis was performed at 200 V for 60 min. For blotting, the proteins in the gel were transferred for 80 min at 100 V in transfer buffer (2.5 mM Tris, 9 mM glycine, 20% methanol) to nitrocellulose membranes (BioRad, Madrid, Spain). Then, membranes were blocked with 8% dry milk and 1% Tween-20 in PBS and incubated with eIF2α-P or α-Actin antibodies, at the recommended dilutions in PBS containing 0.5% dry milk and 0.5% Tween-20 at 4ºC and overnight. Incubation with secondary antibody GAR-Po (Sigma-Aldrich) was done in 0.5% milk 0.5% Tween-20 in PBS for 45 min. Membranes were washed 3 times with PBS containing 1% dry milk 0.5% Tween-20 for 15 min after every antibody incubation. Finally, the membrane was washed 3 times with PBS before analysis of the peroxidase activity. Peroxidase activity was detected using ECL chemiluminescence reagents (Amersham Biosciences, Buckinghamshire, UK) and revealed by exposure to X-ray. Protein bands from western blotting were analysed by densitometry using the Scion Image 4.0.2 Software (RRID: SCR_008673) (www.scionorg.com).\n\nRBCs were fixed with 4% paraformaldehyde (PFA; Sigma-Aldrich) and 0.08% glutaraldehyde (Sigma-Aldrich) diluted in RPMI medium for 20 minutes. Then, RBCs were incubated with permeabilization buffer containing 0.05% saponin (Sigma-Aldrich) in RPMI, for 15 minutes. Primary antibodies were used at 1/50 dilution for IL-1β, IL-8 and TNF-α, 1/300 for Mx and 1/500 for 2F12 in permeabilization buffer and incubated for 60 minutes at room temperature. Secondary antibodies were incubated for 30 minutes at 1/200 dilution. RBCs were washed with permeabilization buffer after antibody incubations. Finally, RBCs were kept in PFA 1% in PBS. For nuclear staining, RBCs were stained with 1 μg/mL of 4′-6-408 Diamidino-2-phenylindole (DAPI; Sigma-Aldrich) for 5 minutes. Flow cytometry (FC) analysis was done in a BD FACSCanto™ II (BD Biosciences) flow cytometer. Immunofluorescence (IF) images were performed in the INCell Analyzer 6000 Cell imaging system (GE Healthcare, Little Chalfont, UK).\n\nConditioned medium (CM) was obtained from control and IPNV-exposed RBCs at MOI 0.5, during 3 days. The CMs were clarified at 1600 rpm for 5 min. To test the antiviral activity of the CM, confluent CHSE-214 cells (7.8×104 cells/well), seeded in 96 well plates were pre-treated with 100 µL of each supernatant at the indicated dilutions for 24 hours. After that, CHSE-214 cells were infected as described previously with IPNV at MOI 0.05, for 24 hours. Finally, intracellular stain of IPNV foci was carried out.\n\nCHSE-214 cells were fixed with PFA diluted at 4% in PBS followed by a second fixation with cold methanol. Each fixation step lasted 15 minutes. Cells were washed with PBS after each fixation step. Blocking buffer containing 5% goat serum (Sigma-Aldrich) and 0.3% Triton X-100 (Sigma-Aldrich) was added to each well for 1 hour. Then, anti-VP3 2F12 antibody was diluted 1/500 in antibody dilution buffer (1% BSA (Sigma-Aldrich), 0.3% Triton X-100) and was incubated for 1 hour. FITC-labelled goat anti-rabbit was used as secondary antibody at 1/300 dilution. Cells were washed three times after each antibody incubation with PBS. IF images were taken INCell Analyzer 6000 imaging system. IN Cell Developer Toolbox 1.9.2 (RRID: SCR_015790; GE Healthcare, Little Chalfont, UK) was used to count number of IPNV foci (positive areas after image segmentation were selected when >21000 fluorescence units and >2500 µm2 criteria was reached).\n\nCell viability was tested using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay30. Briefly, 25 µl of MTT at a final concentration of 1.9 mg/mL were added to previously treated CHSE-214 cells monolayers, seeded in 96 well plates. Cells were incubated for 3 hours at 21ºC with the reagent. Then, the medium was removed from the wells. Formazan crystals were dissolved in 100 µl of 100% DMSO, incubated for 30 minutes. Absorbance was read at 570 nm in the EONTM microplate spectrophotometer (Biotek, Winooski, VT).\n\nAll the figures and graphics show the mean and standard deviation of the data. P-values associated with each graphic are represented by the legends: , p-value < 0.05; , p-value < 0.01; , p-value < 0.001, *, p-value < 0.0001. Graphpad Prism 6 (RRID: SCR_002798, www.graphpad.com) (Graphpad Software Inc., San Diego, A) was used to graphic representation and statistical calculus. FC data were analyzed using Flowing Software 2.5.1 (RRID: SCR_015781)(www.flowingsoftware.com) to obtain Mean Fluorescence Intensity (MFI) and Mean Relative Fluorescence Intensity (MRFI) (relative to control cells) values.\n\nMethodology was carried out in accordance with the Spanish Royal Decree RD 53/2013 and EU Directive 2010/63/EU for animals used in research experimentation. All experimental protocols involving animal handling were also reviewed and approved by the Animal Welfare Body and the Research Ethics Committee at the Miguel Hernandez University (approval number 2014.205.E.OEP; 2016.221.E.OEP) and performed by qualified research personnel.\n\n\nResults\n\nTo evaluate the infectivity of IPNV in trout RBCs, RBCs were exposed to IPNV at MOI 0.5 and the viral RNA was evaluated by RT-qPCR in the cell pellet at different times post-exposure. IPNV infectivity was also evaluated in parallel in the CHSE-214 cell line, used as a positive control of infection. IPNV segment A (IPNV-A) RNA levels inside RBCs and CHSE-214 cell line were similar at 1 and 3 hours post-exposure (hpe) (Figure 1A). After 3 hpe, IPNV-A RNA level was 4 logarithms lower in RBCs in comparison with CHSE-214 cells. On the other hand, the titer of IPNV in the supernatants from IPNV-exposed RBCs at a MOI of 0.5 and 5, was evaluated by TCID50, at 3 days post-exposure (dpe), and showed a recovered titer of 5 and 4 logarithms lower, respectively (Figure 1B). Furthermore, the supernatants titrated by RT-qPCR, were below the lowest limit of detection 102 TCID50 (Table 2). Moreover, there was not significant detection of the IPNV VP3 protein inside RBCs at 3 dpe, by means of flow cytometry (FC) (Figure 1C and D).\n\n(A) Time-course experiment of the expression of IPNV segment A (IPNV-A) in RBCs (●) (n = 6) and CHSE-214 cells (◌) (n = 2) at MOI 0.5. Data is represented as mean±SD. Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all time-points post-exposure in comparison with control time point (0 hpi) (, p-value < 0.05). (B) Recovered virus titer in supernatants from IPNV-exposed RBCs with an inoculum titer of 106 (MOI 0.5) and 107 (MOI 5) TCID50/mL obtained after 72 hpe (n = 5). Data is represented as mean±SD. Mann-Whitney test was performed among both conditions (, p-value < 0.05). (C) MFI (mean fluorescence intensity) of viral protein VP3 in control and IPNV-exposed RBCs at MOI 0.5 and 3 dpe (n = 6) Mann-Whitney test was performed among both conditions. (D) Representative flow cytometry histograms of IPNV VP3 protein detection in control and IPNV-exposed RBCs at MOI 0.5 and 3 dpi.\n\nCt value ± standard deviation from standard line points (108 to 102 dilutions) and supernatans from IPNV-exposed RBCs at MOI 0.5, at 3 and 6 dpe. (n=7 individuals).\n\nThe expression levels of ifn1 and the IFN-1 related genes tlr3, irf7, mx1–3 and pkr, were tested in trout RBCs to determine if they could develop an interferon-related antiviral immune response against IPNV exposure, despite IPNV did not infect trout RBCs. The results showed that mx1–3 and pkr genes were significantly expressed at 72 hpe. On the other hand, ifn1 gene presented a tendency to increase its expression after 6 hpe having a peak at 24 hpe. Also, tlr3 gene expression tended to be upregulated at 24 hpe whereas irf7 expression was upregulated at 72 hpe (Figure 2A). Three and six dpe with IPNV, RBCs were stained intracellularly with an anti-Mx antibody and analyzed by FC and immunofluorescence imaging (IF). The results showed a significant increment in the expression of Mx protein at 6 dpe by both FC an IF (Figure 2B and D). FC histograms showed, at 6 dpe, that RBCs depicted distinct peaks of Mx expression, showing that the expression of Mx in RBCs was heterogeneous in the total RBCs population (Figure 2C).\n\n(A) Gene expression of tlr3, irf7, inf1, mx1–3 and pkr in IPNV-exposed RBCs at the indicated times post-infection and MOI 0.5, measured by RT-qPCR. Data represesent mean±SD (n = 6). Kruskal-Wallis Test with Dunn´s Multiple Comparison post-hoc test was performed among all time-points post-exposure in comparison with control time point (0 hpi) (, p-value < 0.05). (B) Mx protein MRFI (mean relative fluorescent intensity, relative to control cells) in IPNV-exposed RBCs at MOI 0.5 (n = 5). (C) Flow cytometry histograms of Mx protein expression from control (grey) and IPNV-exposed (red) RBCs at MOI 0.5 and the indicated days post-exposure (dpe). (D) Representative immunofluorescence images of Mx protein expression in control and IPNV-exposed RBCs at MOI 0.5 (FITC – Mx protein expression, DAPI - Nuclei) (IF representative of 40 images).\n\nTo analyze if IPNV-exposed RBCs could secrete factors that were capable to protect other fish cells against IPNV infection, conditioned medium (CM) from control and IPNV-exposed RBCs (with IPNV titer <10 TCID50/mL) were added to CHSE-214 cells prior to infection. Figure 3A shows a significant decrease in the number of IPNV infective focus forming units (FFU/mL) when pre-treating with 1/5 diluted CM from IPNV-exposed RBCs. CHSE-214 cells viability, by means of an MTT colorimetric assay, was not affected by the exposure to CM (Figure 3B).\n\n(A) Viral titers (FFU/mL) in CHSE-214 cells infected with IPNV at MOI 0.05 previously non-treated (black) or treated with either supernatans from control RBCs (white) or IPNV-exposed RBCs (grey), during 24 hours, at the indicated dilutions (n = 4, performing triplicates from each individual). Two-way ANOVA test was performed among the different dilutions and conditions to test statistical differences. (B) Percentage of viable CHSE-214 cells pre-treated with conditioned medium from control and IPNV-exposed RBCs, during 24 hours, and relative to non-treated CHSE-214 cells. Percentage of viable cells was calculated as follows: absorbance treated cells/absorbance non-treated cells) x100.\n\nTo evaluate whether ex vivo trout RBCs could produce cytokines, in response to IPNV exposure, that could be involved in the protection conferred by their supernatants, RBCs were exposed to IPNV and IL-1 β, IL-8 and TNF-α protein levels were evaluated by means of FC and IF in control and IPNV-exposed cell cultures. The results showed a decrease in the protein expression of IL-1β, IL-8 and TNFα in IPNV-exposed RBCs (Figure 4A).\n\n(A) Intracellular MFI values of IL-1β, IL-8 and TNFα from control and IPNV-exposed RBCs at MOI 0.5 and 3 dpe measured by FC (n = 6). Mann-Whitney test was performed among both conditions. (B) Phosphorylation of translation initiation factor eIF2α in IPNV-exposed RBCs. Representative western blot of eIF2α-P in control and IPNV-exposed RBCs from two individuals at MOI 0.5, 3 dpe. Densitometry ratios were done relativizing to α-actin. Mann-Whitney test was performed among both conditions.\n\nThe phosphorylation of the translation initiation factor eIF2α is a key mechanism of global inhibition of translational initiation31 and it has been described to happen after IPNV infection in the permissive cell line CHSE-214 cells32. In this sense, since IPNV-exposed RBCs depicted a small downregulation of the evaluated cytokines protein levels, we further investigated whether IPNV exposure could reduce protein translation in RBCs by triggering the phosphorylation of eIF2α. However, the results revealed no changes in the phosphorylation of eIF2α (Figure 4B).\n\n\nDiscussion\n\nPreviously, we have demonstrated that trout RBCs can respond to VHSV, a ssRNA virus not targeting RBCs, halting its replication, downregulating type I interferon-related genes, showing global protein downregulation in the cell and phosphorylation of the translation initiation factor eIF2α (unpublished report, Nombela I, Puente-Marin S, Chico V, Villena A, Carracedo B, Ciordia S, Mena M, Mercado L, Perez L, Coll J, Estepa A, Ortega-Villaizan M).\n\nIt is known that IPNV primarily targets pancreatic and liver cells33. It has been also reported that IPNV was detectable in kidney hematopoietic tissue, corpuscles of Stannius, in Islets of Langherhans, in the lamina propria of the pyloric caeca, the gill arch connective tissue, the ventricle of the heart and dermis of the skin33. Our results showed that IPNV did not replicate in RBCs, although small amounts of IPNV were persistently found inside RBCs after 3 dpe (≈ 103 TCID50/mL). Similarly, IPNV has been shown to enter mammalian cells, without significant levels of replication, being this entry suggested to be receptor mediated34. From our results, the persistence of IPNV in RBCs after 72 hpe could point out the entry of the virus inside RBCs. However, we could not verify the presence of the IPNV inside RBCs\n\nNevertheless, despite the lack of replication of IPNV in RBCs, IPNV could induce an antiviral gene expression mediated by the IFN pathway, as it has been observed in RBCs productive infections with ISAV8 and PRV9. As shown by our results, ifn1 and IFN-1 related genes (irf7, pkr and mx) expression levels were increased time-dependently in response to IPNV-exposure. High inter-individual variability was observed, similarly to that found in the RBCs from salmons challenged with PRV35. In addition, although we could not verify the entry and uncoating of IPNV inside RBCs, we could observe an increment in the expression of the tlr3 gene in parallel to the expression of the other IFN-related genes in IPNV-exposed RBCs. This could indicate the activation of the TLR3/IFN pathway by the presence of intracellular viral dsRNA.\n\nIFN-1 leads to the expression of interferon stimulated genes (ISGs)36. Among ISGs, the antiviral protein Mx has a well characterized antiviral role. Confirming those expectations, our results showed the significant upregulation of the Mx protein 6 dpe, after having a peak of its gene expression at 3 dpe. Previously, a positive correlation between the expression of Mx protein and the inhibition of IPNV in CHSE-214 cells has been established37. Therefore, Mx protein production in IPNV-exposed RBCs could be involved in the low IPNV titers observed. The high basal levels of Mx protein detected inside RBCs, much elevated than those for CHSE-214 cells (Figure S1), could be implicated in the early disappearance of IPNV inside RBCs. A similar hypothesis has been made in the abortive infection of VHSV in the RTS-11 cell line38 and in trout RBCs (unpublished report, Nombela I, Puente-Marin S, Chico V, Villena A, Carracedo B, Ciordia S, Mena M, Mercado L, Perez L, Coll J, Estepa A, Ortega-Villaizan M), where upregulation or high constitutive expression of mx genes was speculated to be related to the inhibition of the virus.\n\nMoreover, our results showed that CM from RBCs exposed to IPNV could partially protect CHSE-214 cells from IPNV infection. Similar to other cell types, this antiviral activity has been also observed in CM of RTS11 and RTG-2 cells exposed to Poly (I:C) (polyinosinic:polycytidylic acid) and/or infected with chum salmon reovirus39. The fact that RBCs can secrete factors that confer protection against IPNV infection in other cell lines could indicate that RBCs, despite not being permissive to IPNV infection, may exhibit an antiviral response. To elucidate which factors could be implicated in this protection, we evaluated the production of cytokines in IPNV-exposed RBCs. Previously, the expression of IL-1β in salmon gill and head kidney tissues40, IL-8 in trout head kidney tissue41 and TNFα in zebrafish embryonic cells42 have been implicated in the immune response against IPNV; therefore, we chose these cytokines to evaluate the immune response of trout RBCs to IPNV exposure. However, our results showed a downregulation of these proteins in IPNV-exposed RBCs.\n\nA shutdown in protein synthesis by phosphorylation of eIF2α has been reported in CHSE-214 cells infected with IPNV32. So far, in trout RBCs, although cytokine protein downregulation was observed, no phosphorylation of eIF2α was detected and Mx protein expression was increased. In contrast, in trout RBCs, VHSV rhabdovirus induced phosphorylation of eIF2α and a cell shut-off characterized by the downregulation of the proteome (unpublished report, Nombela I, Puente-Marin S, Chico V, Villena A, Carracedo B, Ciordia S, Mena M, Mercado L, Perez L, Coll J, Estepa A, Ortega-Villaizan M). On the other hand, IFN-1 has been reported to inhibit the production of IL-1β43.\n\nFurther studies are needed to completely understand the molecular mechanism through which IPNV triggers this immune response in trout RBCs. However, the lack of commercial antibodies against fish proteins involved in cell signaling networks limits the study of this area. The implication of RBCs during in vivo IPNV infection and the response against different strains of IPNV remains to be evaluated.\n\nFinally, one of the potential applications of these results is that fish RBCs could be considered mediators of the antiviral response and therefore targets of novel DNA vaccines and of new strategies against fish viral infections.\n\n\nData availability\n\nDataset 1. Excel file containing qPCR data. Each sheet contains the raw Ct values for the indicated figure numbers, organized by samples (rows) and genes (columns). doi, 10.5256/f1000research.12994.d18284249\n\nDataset 2. Excel file containing the virus titration data. Contains the virus titer (TCID50/mL) results of the indicated figure number. doi, 10.5256/f1000research.12994.d18284350\n\nDataset 3. Flow cytometry data. Each folder contains the Flow Cytometry Standard (.fcs) format files. Source data files are organized by figure number, and then by sample number, condition and antibody. doi, 10.5256/f1000research.12994.d18284451\n\nDataset 4. Excel file containing the Focus Forming Units (FFU) counting for Figure 3A. doi, 10.5256/f1000research.12994.d18284552\n\nDataset 5. Excel file containing MTT absorbance raw data. doi, 10.5256/f1000research.12994.d18284653\n\nDataset 6. Excel file containing the densitometry raw data of eIF2α-P and α-Actin western blots. Related uncropped blots are included. doi, 10.5256/f1000research.12994.d18284754", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the European Research Council (ERC starting grant 2014 GA639249).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nSpecial thanks to Remedios Torres and Efren Lucas for their technical assistance.\n\n\nSupplementary material\n\nFigure S1. qPCR Amplification plot of mx1-3 gene expression in RBCs and CHSE-214 cells.\n\nClick here to access the data.\n\n\nReferences\n\nWolf K, Snieszko SF, Dunbar CE, et al.: Virus nature of infectious pancreatic necrosis in trout. Proc Soc Exp Biol Med. 1960; 104: 105–8. PubMed Abstract \| Publisher Full Text\n\nChristie KE, Ness S, Djupvik HO: Infectious pancreatic necrosis virus in Norway: partial serotyping by monoclonal antibodies. J Fish Dis. 1990; 13(4): 323–7. Publisher Full Text\n\nZhu L, Wang X, Wang K, et al.: Outbreak of infectious pancreatic necrosis virus (IPNV) in farmed rainbow trout in China. Acta Trop. 2017; 170: 63–9. PubMed Abstract \| Publisher Full Text\n\nSnieszko SF: Your fishes’ health: Diseases of fishes. Trop Fish Hobbyist. 1981; 30(3). Reference Source\n\nDobos P, Hill BJ, Hallett R, et al.: Biophysical and biochemical characterization of five animal viruses with bisegmented double-stranded RNA genomes. J Virol. 1979; 32(2): 593–605. PubMed Abstract \| Free Full Text\n\nDobos P: The molecular biology of infectious pancreatic necrosis virus (IPNV). Ann Rev Fish Dis. 1995; 5: 25–54. Publisher Full Text\n\nCaswell-Reno P, Reno PW, Nicholson BL: Monoclonal antibodies to infectious pancreatic necrosis virus: analysis of viral epitopes and comparison of different isolates. J Gen Virol. 1986; 67(Pt 10): 2193–205. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRodriguez MF, Wiens GD, Purcell MK, et al.: Characterization of Toll-like receptor 3 gene in rainbow trout (Oncorhynchus mykiss). Immunogenetics. 2005; 57(7): 510–9. PubMed Abstract \| Publisher Full Text\n\nPassantino L, Massaro MA, Jirillo F, et al.: Antigenically activated avian erythrocytes release cytokine-like factors: a conserved phylogenetic function discovered in fish. Immunopharmacol Immunotoxicol. 2007; 29(1): 141–52. PubMed Abstract \| Publisher Full Text\n\nPassantino L, Altamura M, Cianciotta A, et al.: Maturation of fish erythrocytes coincides with changes in their morphology, enhanced ability to interact with Candida albicans and release of cytokine-like factors active upon autologous macrophages. Immunopharmacol Immunotoxicol. 2004; 26(4): 573–85. PubMed Abstract \| Publisher Full Text\n\nPassantino L, Altamura M, Cianciotta A, et al.: Fish immunology. I. Binding and engulfment of Candida albicans by erythrocytes of rainbow trout (Salmo gairdneri Richardson). Immunopharmacol Immunotoxicol. 2002; 24(4): 665–78. PubMed Abstract \| Publisher Full Text\n\nSchraml B, Baker MA, Reilly BD: A complement receptor for opsonized immune complexes on erythrocytes from Oncorhynchus mykiss but not Ictalarus punctatus. Mol Immunol. 2006; 43(10): 1595–603. PubMed Abstract \| Publisher Full Text\n\nBeck Z, Brown BK, Wieczorek L, et al.: Human erythrocytes selectively bind and enrich infectious HIV-1 virions. PLoS One. 2009; 4(12): e8297. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHill BJ, Way K: Serological classification of infectious pancreatic necrosis (IPN) virus and other aquatic birnaviruses. Annu Rev Fish Dis. 1995; 5: 55–77. Publisher Full Text\n\nMarroquí L, Estepa A, Perez L: Inhibitory effect of mycophenolic acid on the replication of infectious pancreatic necrosis virus and viral hemorrhagic septicemia virus. Antiviral Res. 2008; 80(3): 332–8. PubMed Abstract \| Publisher Full Text\n\nChico V, Gomez N, Estepa A, et al.: Rapid detection and quantitation of viral hemorrhagic septicemia virus in experimentally challenged rainbow trout by real-time RT-PCR. J Virol Methods. 2006; 132(1–2): 154–9. PubMed Abstract \| Publisher Full Text\n\nLivak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001; 25(4): 402–8. PubMed Abstract \| Publisher Full Text\n\nRojas V, Camus-Guerra H, Guzmán F, et al.: Pro-inflammatory caspase-1 activation during the immune response in cells from rainbow trout Oncorhynchus mykiss (Walbaum 1792) challenged with pathogen-associated molecular patterns. J Fish Dis. 2015; 38(11): 993–1003. PubMed Abstract \| Publisher Full Text\n\nSchmitt P, Wacyk J, Morales-Lange B, et al.: Immunomodulatory effect of cathelicidins in response to a β-glucan in intestinal epithelial cells from rainbow trout. Dev Comp Immunol. 2015; 51(1): 160–9. PubMed Abstract \| Publisher Full Text\n\nSantana P, Palacios C, Narváez E, et al.: Anti-peptide antibodies: A tool for detecting IL-8 in salmonids. Electron J Biotechnol. 2012; 15(5). Publisher Full Text\n\nRojas V, Morales-Lange B, Guzmán F, et al.: Immunological strategy for detecting the pro-inflammatory cytokine TNF-alpha in salmonids. Electron J Biotechnol. 2012; 15(5). Publisher Full Text\n\nChico V, Martinez-Lopez A, Ortega-Villaizan M, et al.: Pepscan mapping of viral hemorrhagic septicemia virus glycoprotein G major lineal determinants implicated in triggering host cell antiviral responses mediated by type I interferon. J Virol. 2010; 84(14): 7140–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMartinez-Lopez A, Garcia-Valtanen P, Ortega-Villaizan M, et al.: VHSV G glycoprotein major determinants implicated in triggering the host type I IFN antiviral response as DNA vaccine molecular adjuvants. Vaccine. 2014; 32(45): 6012–9. PubMed Abstract \| Publisher Full Text\n\nDomínguez J, Hedrick RP, Sánchez-Vizcaino JM: Use of monoclonal-antibodies for detection of infectious pancreatic necrosis virus by the enzyme-linked-immunosorbent-assay (ELISA). Dis Aquat Organ. 1990; 8: 157–63. Publisher Full Text\n\nRoehm NW, Rodgers GH, Hatfield SM, et al.: An improved colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT. J Immunol Methods. 1991; 142(2): 257–65. PubMed Abstract \| Publisher Full Text\n\nLevin D, London IM: Regulation of protein synthesis: activation by double-stranded RNA of a protein kinase that phosphorylates eukaryotic initiation factor 2. Proc Natl Acad Sci U S A. 1978; 75(3): 1121–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGamil AA, Mutoloki S, Evensen Ø: A piscine birnavirus induces inhibition of protein synthesis in CHSE-214 cells primarily through the induction of eIF2α phosphorylation. Viruses. 2015; 7(4): 1987–2005. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEllis AE, Cavaco A, Petrie A, et al.: Histology, immunocytochemistry and qRT-PCR analysis of Atlantic salmon, Salmo salar L., post-smolts following infection with infectious pancreatic necrosis virus (IPNV). J Fish Dis. 2010; 33(10): 803–18. PubMed Abstract \| Publisher Full Text\n\nØrpetveit I, Küntziger T, Sindre H, et al.: Infectious pancreatic necrosis virus (IPNV) from salmonid fish enters, but does not replicate in, mammalian cells. Virol J. 2012; 9: 228. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDahle MK, Wessel Ø, Timmerhaus G, et al.: Transcriptome analyses of Atlantic salmon (Salmo salar L.) erythrocytes infected with piscine orthoreovirus (PRV). Fish Shellfish Immunol. 2015; 45(2): 780–90. PubMed Abstract \| Publisher Full Text\n\nSchoggins JW, Rice CM: Interferon-stimulated genes and their antiviral effector functions. Curr Opin Virol. 2011; 1(6): 519–25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLarsen R, Røkenes TP, Robertsen B: Inhibition of infectious pancreatic necrosis virus replication by atlantic salmon Mx1 protein. J Virol. 2004; 78(15): 7938–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPham PH, Lumsden JS, Tafalla C, et al.: Differential effects of viral hemorrhagic septicaemia virus (VHSV) genotypes IVa and IVb on gill epithelial and spleen macrophage cell lines from rainbow trout (Oncorhynchus mykiss). Fish Shellfish Immunol. 2013; 34(2): 632–40. PubMed Abstract \| Publisher Full Text\n\nDeWitte-Orr SJ, Leong JA, Bols NC: Induction of antiviral genes, Mx and vig-1, by dsRNA and Chum salmon reovirus in rainbow trout monocyte/macrophage and fibroblast cell lines. Fish Shellfish Immunol. 2007; 23(3): 670–82. PubMed Abstract \| Publisher Full Text\n\nIngerslev HC, Rønneseth A, Pettersen EF, et al.: Differential expression of immune genes in Atlantic salmon (Salmo salar L.) challenged intraperitoneally or by cohabitation with IPNV. Scand J Immunol. 2009; 69(2): 90–8. PubMed Abstract \| Publisher Full Text\n\nReyes-Cerpa S, Reyes-López F, Toro-Ascuy D, et al.: Induction of anti-inflammatory cytokine expression by IPNV in persistent infection. Fish Shellfish Immunol. 2014; 41(2): 172–82. PubMed Abstract \| Publisher Full Text\n\nWang WL, Liu W, Gong HY, et al.: Activation of cytokine expression occurs through the TNFα/NF-κB-mediated pathway in birnavirus-infected cells. Fish Shellfish Immunol. 2011; 31(1): 10–21. PubMed Abstract \| Publisher Full Text\n\nGuarda G, Braun M, Staehli F, et al.: Type I interferon inhibits interleukin-1 production and inflammasome activation. Immunity. 2011; 34(2): 213–23. PubMed Abstract \| Publisher Full Text\n\nRaida MK, Buchmann K: Temperature-dependent expression of immune-relevant genes in rainbow trout following Yersinia ruckeri vaccination. Dis Aquat Organ. 2007; 77(1): 41–52. PubMed Abstract \| Publisher Full Text\n\nOrtega-Villaizan M, Chico V, Martinez-Lopez A, et al.: In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells. Vaccine. 2011; 29(4): 737–43. PubMed Abstract \| Publisher Full Text\n\nWang T, Bird S, Koussounadis A, et al.: Identification of a novel IL-1 cytokine family member in teleost fish. J Immunol. 2009; 183(2): 962–74. PubMed Abstract \| Publisher Full Text\n\nWang T, Diaz-Rosales P, Costa MM, et al.: Functional characterization of a nonmammalian IL-21: rainbow trout Oncorhynchus mykiss IL-21 upregulates the expression of the Th cell signature cytokines IFN-gamma, IL-10, and IL-22. J Immunol. 2011; 186(2): 708–21. PubMed Abstract \| Publisher Full Text\n\nPurcell MK, Nichols KM, Winton JR, et al.: Comprehensive gene expression profiling following DNA vaccination of rainbow trout against infectious hematopoietic necrosis virus. Mol Immunol. 2006; 43(13): 2089–106. PubMed Abstract \| Publisher Full Text\n\nNombela I, Carrion A, Puente-Marin S, et al.: Dataset 1 in: Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective. F1000Research. 2017. Data Source\n\nNombela I, Carrion A, Puente-Marin S, et al.: Dataset 2 in: Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective. F1000Research. 2017. Data Source\n\nNombela I, Carrion A, Puente-Marin S, et al.: Dataset 3 in: Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective. F1000Research. 2017. Data Source\n\nNombela I, Carrion A, Puente-Marin S, et al.: Dataset 4 in: Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective. F1000Research. 2017. Data Source\n\nNombela I, Carrion A, Puente-Marin S, et al.: Dataset 5 in: Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective. F1000Research. 2017. Data Source\n\nNombela I, Carrion A, Puente-Marin S, et al.: Dataset 6 in: Piscine birnavirus triggers antiviral immune response in trout red blood cells, despite not being infective. F1000Research. 2017. Data Source" }	[ { "id": "27699", "date": "20 Nov 2017", "name": "Espen Rimstad", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting paper showing that purified rainbow trout RBC exposed to IPNV do not get infected by the virus, but nevertheless raise an innate antiviral response. The latter is shown by the induction of IFN and a few IFN related genes and that conditioned media from IPNV exposed RBC inhibits IPNV infection of the susceptibe cell line CHSE-214. Many methods are used to approach the hypotheses and the results appear provide new information on the very intriguing role of fish red blood cells in interaction with viruses, in particular by showing that the cells can induce an antiviral immune response without being infected. The main comments are related to how the authors have interpreted the results far beyond what they have shown, by drawing links to adaptive immune mechanisms and vaccination approaches, while they could have discussed antiviral protection mechanisms in further detail and with more scientific basis. There are also some missing information regarding the experimental details, and far too extensive use of an unpublished paper in the discussion of the data.\n\nComments:\nThe title is generalizing beyond the scope of the paper, and should specify the virus and the host studied (IPNV and onchorhynchus mykiss). Abstract and Discussion: The statement that antiviral responses in RBCs could be targets for DNA vaccines is not linked to the data presented here. A vaccination effect requires antigen presentation and adaptive immune activation for long term protection, which is not touched upon at all in this paper. If this point should be made, the authors should in the discussion provide some explanation and scientific basis for how they think antiviral responses in RBCs to an uninfecting virus make them promising DNA vaccination targets, and how directing DNA vaccines to RBC should be performed. Introduction/Discussion: An unpublished paper: Nombela I, Puente-Marin S et al. is referred to in introduction and several times in the discussion. The content of this paper cannot be evaluated. The authors should primarily use published work in their discussion. Introduction. There are many language mistakes in the introduction (and elsewhere). Please have the manuscript corrected linguistically. Trout is not a specific annotation. Please specify that the work is performed for rainbow trout. Diseases are not agents. Diseases cause losses not loses. Viral titer not titters. Aquabirnavirus genus not Aquabirnaviridae genera, it is wrong suffix and it is not plural. etc. The role of IPNV - VP3 is not completely unknown, there are several papers describing its function. “The VP2 epitopes are the target for antibodies against different serotypes.” Not against but distinguishing between. It should be noted that the serotyping system is based upon the use of rabbit antisera, it is not based on the immune response from fish. No observations of viral factories has been observed for ISAV. “The fact that these cells are nucleated implies that fish RBCs can respond to different stimulus and control cellular processes”. Not really, having a nucleus doesn’t really prove that the cell can react various stimuli, that have to shown. Materials and Methods: Number of fish used not given. Unclear if the fish used were tested for previous viral infections. 18S is given as the reference gene target in the text, but in the table EF1a primers are given. Results is not clear on what was used. Results: Unclear if the “n” in experiments is number of fish or parallel ex vivo infections. “IPNV segment A (IPNV-A) RNA levels inside RBCs and CHSE-214 cell line were similar at 1 and 3 hours post-exposure (hpe) (Figure 1A). After 3 hpe, IPNV-A RNA level was 4 logarithms lower in RBCs in comparison with CHSE-214 cells.” Do you mean from 6 hpe and not after 3 hpe? It is not 4 logs difference according to Fig 1 it is 3 log at 6 hpe and 5 log at 26 hpe. It is nevenr 4 logs? What is the detection limit for VP3 in flow cytometri? Discussion:\nThe small trend towards a difference in cytokine levels Fig 4A should not be discussed as “downregulation”, and cannot be interpreted as a cell shut-off of the proteome by the infection. Most likely the slight trend towards down-regulation is just an effect of the relative upregulation of the IFN-I pathway (ISGs) Authors say in the discussion regarding the protective effect of conditioned media: “To elucidate which factors could be implicated in this protection, we evaluated the production of cytokines in IPNV-exposed RBCs” It is well known that IFN-I, and not to the same degree cytokines, confers a direct antiviral response on surrounding cells. The upregulation of IFN-I is already shown at the transcriptional level, and the upregulation of ISGs directly indicates IFN activity. The main role of cytokines is to attract and activate immune cells and mobilize an immune response in tissue in vivo. The authors should clarify this to show that they understand the difference between local antiviral protection and crosstalk with the immune system.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3220", "date": "13 Dec 2017", "name": "Maria del Mar Ortega-Villaizan Romo", "role": "Author Response", "response": "Dear Dr. Espen Rimstad and Dr. Maria Dahle, We appreciate very much your revision and constructive comments on the manuscript. We have included your corrections in the new version of the manuscript hoping that now the manuscript will be suitable for publication. Please find below the response to your comments:1. The title is generalizing beyond the scope of the paper, and should specify the virus and the host studied (IPNV and onchorhynchus mykiss).Author’s response: The title have been changed as advised.2. Abstract and Discussion: The statement that antiviral responses in RBCs could be targets for DNA vaccines is not linked to the data presented here. A vaccination effect requires antigen presentation and adaptive immune activation for long term protection, which is not touched upon at all in this paper. If this point should be made, the authors should in the discussion provide some explanation and scientific basis for how they think antiviral responses in RBCs to an uninfecting virus make them promising DNA vaccination targets, and how directing DNA vaccines to RBC should be performed.Author’s response: In order to avoid misunderstanding we have deleted DNA vaccines from abstract3. Introduction/Discussion: An unpublished paper: Nombela I, Puente-Marin S et al. is referred to in introduction and several times in the discussion. The content of this paper cannot be evaluated. The authors should primarily use published work in their discussion.Author’s response: The paper reference can be included now and has been included.4. Introduction. There are many language mistakes in the introduction (and elsewhere). Please have the manuscript corrected linguistically. Trout is not a specific annotation.Author’s response: the manuscript has been linguistically corrected and trout has been properly annotated as rainbow trout. 5. Please specify that the work is performed for rainbow trout. Diseases are not agents. Diseases cause losses not loses. Viral titer not titters. Aquabirnavirus genus not Aquabirnaviridae genera, it is wrong suffix and it is not plural. etc.Author’s response: we have specified that the work has been done for rainbow trout. Titer has been written instead of titters. Aquabirnavirus genus has been written instead of Aquabirnaviridae genera.6. The role of IPNV - VP3 is not completely unknown, there are several papers describing its function.Author’s response: This sentence has been deleted since it is not the target of our study.7. “The VP2 epitopes are the target for antibodies against different serotypes.” Not against but distinguishing between. It should be noted that the serotyping system is based upon the use of rabbit antisera, it is not based on the immune response from fish.Author’s response: This sentence has been deleted since it is not the target of our study.8. No observations of viral factories has been observed for ISAV.Author’s response: We have deleted that sentence.9. “The fact that these cells are nucleated implies that fish RBCs can respond to different stimulus and control cellular processes”. Not really, having a nucleus doesn’t really prove that the cell can react various stimuli, that have to shown.Author’s response: We have deleted that sentence.10. Materials and Methods: Number of fish used not given. Unclear if the fish used were tested for previous viral infections.Author’s response: The number of fish is indicated in each experiment/figure. On the other hand, the fish has not been tested for previous infection. However, the fish farm appears to be free of IPNV and VHSV, since they do not have associated mortalities. 11. 18S is given as the reference gene target in the text, but in the table EF1a primers are given. Results is not clear on what was used.Author’s response: Yes, this has been an error. EF1a has been removed from the primers table.12. Results: Unclear if the “n” in experiments is number of fish or parallel ex vivo infections.Author’s response: Yes, “n” refers to number of fish/ ex vivo infections13. “IPNV segment A (IPNV-A) RNA levels inside RBCs and CHSE-214 cell line were similar at 1 and 3 hours post-exposure (hpe) (Figure 1A). After 3 hpe, IPNV-A RNA level was 4 logarithms lower in RBCs in comparison with CHSE-214 cells.” Do you mean from 6 hpe and not after 3 hpe? It is not 4 logs difference according to Fig 1 it is 3 log at 6 hpe and 5 log at 26 hpe. It is nevenr 4 logs?Author’s response: Yes, please excuse the error. We have corrected it as “After 6 hpe, IPNV-A RNA level was 3 logarithms lower”14. What is the detection limit for VP3 in flow cytometri?Author’s response: We do not know the limit of detection for VP3 by flow cytometry. However, by ELISA, it has been described that the limit of the detection for 2F12 + 3B12 antibody mix (anti-VP3) is 104 TCID50/ml (Domínguez J, Hedrick RP, Sánchez-Vizcaino JM: Use of monoclonal-antibodies for detection of infectious pancreatic necrosis virus by the enzyme-linked-immunosorbent-assay (ELISA). Dis Aquat Organ. 1990; 8: 157–63. Publisher Full Text).15. Discussion:1. The small trend towards a difference in cytokine levels Fig 4A should not be discussed as “downregulation”, and cannot be interpreted as a cell shut-off of the proteome by the infection. Most likely the slight trend towards down-regulation is just an effect of the relative upregulation of the IFN-I pathway (ISGs)Author’s response: We have included this appreciation in the discussion.2. Authors say in the discussion regarding the protective effect of conditioned media: “To elucidate which factors could be implicated in this protection, we evaluated the production of cytokines in IPNV-exposed RBCs” It is well known that IFN-I, and not to the same degree cytokines, confers a direct antiviral response on surrounding cells. The upregulation of IFN-I is already shown at the transcriptional level, and the upregulation of ISGs directly indicates IFN activity. The main role of cytokines is to attract and activate immune cells and mobilize an immune response in tissue in vivo. The authors should clarify this to show that they understand the difference between local antiviral protection and crosstalk with the immune system. Author’s response: In order to avoid misunderstandings, and due to lack of information about il8, il1b and tnfa receptor pathways in CHSE-214, we have deleted in the manuscript the relation between il8, il1b and tnfa with the protection of CHSE against IPNV." } ] }, { "id": "27697", "date": "23 Nov 2017", "name": "Niels C. Bols", "expertise": [ "Reviewer Expertise Fish cell line development", "fish virology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript by Maria de Mar Ortega-Villaizan Romo provides evidence that trout red blood cells can express antiviral mechanisms. The discovery is very intriguing, as red blood cells appear to have addition functions beyond carry gases, and this might open up new ways to enhance fish health. Therefore, I recommend indexing. As documented below, I do have many minor suggestions for improving the clarity and aesthetics of the manuscript.\nABSTRACT Background Remove “highly” I suggest “However, the roles of RBCs in birnavirus….” Methods: Remove “induced” Results: Remove “-exposed RBCs” Conclusions: I suggest recasting a few sentences as shown below. “Despite not being infected, trout RBCs respond to IPNV with increased expression of antiviral genes.” In the last sentence, I suggest “that triggers this antiviral response in trout RBCs”\nINTRODUCTION 1st paragraph 2nd line remove “Among all of the viral diseases,” It sounds awkward and the point being made is made again in the 4th paragraph. 2nd paragraph Remove “This implies that nucleated RBCs can support viral replication.” Over 40 years ago, enucleated cells were shown to support replication of some viruses1. Therefore, having a nucleus in RBCs might have nothing to do with their capacity to support the replication of some viruses. For example, human RBCs might also have roles in viral infections. Therefore, the first 2 sentences of this paragraph could be eliminated and the next sentence, after removing “for example”, would become the topic sentence.\n\n3rd paragraph Remove the sentence that begins “Recently, it has been shown that viral hemorrhagic septicemia virus (VHSV) can induce ….” I don’t see how it fits the subject of the paragraph or point out in the sentence that RBCs as well as stromal cells were studied. 4th paragraph recast the last 2 sentences “However, we report that even though not infecting RBCs, IPNV induces in them the expression of antiviral genes in the INF-1 pathway.” I am not sure that I would emphasize so strongly “protection against IPNV infection in CHSE-214 cells”. This appears to be based on the 1/5 bar in Figure 3A. I would like to have seen if protection also arose from 1 to 3 supernatant dilution or 1 to 10 supernatant dilution. If either one or both of these dilutions did protect, it would strongly indicate that protection can be repeatability demonstrated.\n\nMETHODS Animals Space between 1 and fish. Space after Spain)\n\nRBC purification Was no anticoagulant used in the collection of the blood? Please clarify. Would thrombocytes be present among the RBCs?\n\nIn the 3rd last sentence I wonder if something should follow the 2%. RPMI with 2 % FBS? This also comes in the next section. Maybe define it here as RPMI with 2 % FBS (RPMI/FBS) and use in RPMI/FBS in subsequent section.\n\nViral infection assays No need to repeat Oncorhynchus mykiss here as the genus species name are mentioned earlier. In the 2nd last sentence, I believe that the RPMI was “removed” rather than “retired”. Were RBC infected at 14 °C like CHSE-214 were?\n\nViral titration assay In the first sentence, “supernatants were quantified by TCID50 and by RT-qPCR.”\n\nRNA isolation and DNAse treatment no suggestions\n\nGene expression by RT-qPCR “synthesized”\n\nAntibodies I suggest the following topic sentence for this section. “Several antibodies were used to stain cells for cytokines and to measure polypeptides in RBC extracts by western blotting and they are briefly described below and their Research Resource Identifiers (RRIDs) given.”\n\nWestern blot no suggestions\n\nIntracellular immunofluorescence staining and flow cytometry\n\nno suggestions\n\nAntiviral activity of conditioned medium Would not the conditioned medium (CM) have virus that had not bound to RBC and thus not pelleted at 1600 rpm and so was in the CM? Please briefly discuss.\n\nIs ‘staining’ rather than “stain” meant in the following sentence and the next heading? “Finally, intracellular stain of IPNV foci was carried out.”\n\nIntracellular stain of IPNV foci Add to the end of the 3rd sentence “for 1 hour with the cells”.\n\nMTT assays At the end of this section, a statement of how the absorbance readings are converted to percent viability should be given. I know that it is mentioned in the legend for Figure 3.\n\nSoftware and statistics I suggest the following modification to the 2nd sentence. “was used for preparing graphs and preforming statistical calculations”.\n\nEthics approval no suggestions\n\nRESULTS IPNV did not infect trout RBCs I suggest the following recasting of the last sentence and addition of a new last sentence. “Moreover, flow cytometry analysis of control and IPNV-exposed RBCs for IPNV VP3 protein did not show significant differences (Figure 1C and D). Therefore, IPNV did not infect trout RBCs.”\n\nIPNV exposure increased the expression of interferon-related antiviral immune genes and proteins in RBCs The first sentence should be shortened as follows “to determine if IPNV would induce an antiviral response in RBCs.” Are there antiviral genes that are not immune genes? In other words, can the description be simplified to just ‘antivrial genes”?\n\nDISCUSSION 2nd paragraph The last two sentences could refer to the data of the ms for support.\n\n4th paragraph I wonder if the authors could briefly discuss the apparent constitutive expression of Mx in RBCs as seen in Panel D of Figure 2. Also could Mx be released from RBCs? In cattle Mx1 is found in exosomes (see Racicot K et al., 2012, Am J Reprod Immunol) and exosomes can be released by RBCs (see Danesh A et al., 2014, Blood).\n\nFIGURES AND LEGENDS\n\nFigure 1 In panel A write out hpe on the X axis.\n\nFigure 2 Is the staining in the control of interest or just background? I mentioned this earlier under the Discussion. Could any of the cells in panel D be thrombocytes?\n\nFigure 3 Could FFU be written out here in the legend so figure stands alone? The legend mentions doing a two-way ANOVA but no mention of a post test is given, although the 1/5 supernatant dilution is identified with an asterisk?\n\nFigure 4 Title legend has a spelling mistake and is a bit misleading because panel B is not covered by the current title. I suggest the following title. “Reduction in RBC cytokine levels by IPNV and a possible mechanism” The legend should give more information so the figure can better stand by itself. Write out MFI (Mean Fluorescence Intensity) and FC (Flow Cytometry). At two places in the legend, mention is made of the Mann-Whitney test but the outcome is not clearly stated.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3219", "date": "13 Dec 2017", "name": "Maria del Mar Ortega-Villaizan Romo", "role": "Author Response", "response": "Dear Dr. Niels Bols,Thank you very much for your positive review and suggestions for the manuscript. We have included your corrections and suggestions in the new version of the manuscript.ABSTRACTBackgroundRemove “highly”Author´s response: It has been corrected.I suggest “However, the roles of RBCs in birnavirus….”Author´s response: It has been corrected.Methods:Remove “induced”Author´s response: It has been corrected.Results:Remove “-exposed RBCs”Author´s response: It has been corrected.Conclusions: I suggest recasting a few sentences as shown below.“Despite not being infected, trout RBCs respond to IPNV with increased expression of antiviral genes.”In the last sentence, I suggest “that triggers this antiviral response in trout RBCs”Author´s response: It has been corrected.INTRODUCTION1st paragraph 2nd line remove “Among all of the viral diseases,”It sounds awkward and the point being made is made again in the 4th paragraph.Author´s response: It has been corrected.2nd paragraphRemove “This implies that nucleated RBCs can support viral replication.” Over 40 years ago, enucleated cells were shown to support replication of some viruses1. Therefore, having a nucleus in RBCs might have nothing to do with their capacity to support the replication of some viruses. For example, human RBCs might also have roles in viral infections. Therefore, the first 2 sentences of this paragraph could be eliminated and the next sentence, after removing “for example”, would become the topic sentence.Author´s response: We have removed these sentences.3rd paragraphRemove the sentence that begins “Recently, it has been shown that viral hemorrhagic septicemia virus (VHSV) can induce ….”I don’t see how it fits the subject of the paragraph or point out in the sentence that RBCs as well as stromal cells were studied.Author´s response: We have changed this sentence to: “Besides, recently it has been shown that viral hemorrhagic septicemia virus (VHSV) halted replication in rainbow trout RBCs could induce cytokine production” as another reference of immune response by RBCs against virus.4th paragraphrecast the last 2 sentences“However, we report that even though not infecting RBCs, IPNV induces in them the expression of antiviral genes in the INF-1 pathway.” I am not sure that I would emphasize so strongly “protection against IPNV infection in CHSE-214 cells”. This appears to be based on the 1/5 bar in Figure 3A. I would like to have seen if protection also arose from 1 to 3 supernatant dilution or 1 to 10 supernatant dilution. If either one or both of these dilutions did protect, it would strongly indicate that protection can be repeatability demonstrated.Author´s response: The protection conferred by the CM from IPNV-exposed RBCs diminished as the dilution increased from 1/5 to 1/25 and 1/125. However, we have not tried 1/3 or 1/10 dilutions. As Dr. Bols pointed out, it would have been interesting to check with lower dilution ranges. In relation to repeatability, this assay was done using the supernatants from 4 ex vivo infections, in triplicate each of them. Therefore, the repeatability could have been demonstrated by the assay itself? We have rewritten the sentence to do not emphasize so strongly “protection” as follows: “the induction of an antiviral state to IPNV in CHSE-214 cells”. METHODSAnimalsSpace between 1 and fish.Space after Spain)Author´s response: It has been corrected.RBC purificationWas no anticoagulant used in the collection of the blood? Please clarify. Would thrombocytes be present among the RBCs?Author´s response: We did not use any anti-coagulant. The blood was immediately diluted in RPMI 10%FBS and slightly resuspended. In this way, no coagulation occurs. On the other hand, thrombocytes were eliminated with the double Ficoll purification.In the 3rd last sentence I wonder if something should follow the 2%. RPMI with 2 % FBS? This also comes in the next section. Maybe define it here as RPMI with 2 % FBS (RPMI/FBS) and use in RPMI/FBS in subsequent section.Author´s response: It has been corrected.Viral infection assaysNo need to repeat Oncorhynchus mykiss here as the genus species name are mentioned earlier.In the 2nd last sentence, I believe that the RPMI was “removed” rather than “retired”.Were RBC infected at 14 °C like CHSE-214 were?Author´s response: It has been corrected and 14 °C added to RBCs infection protocol.Viral titration assayIn the first sentence, “supernatants were quantified by TCID50 and by RT-qPCR.”Author´s response: It has been corrected. RNA isolation and DNAse treatmentno suggestions Gene expression by RT-qPCR“synthesized”Author´s response: It has been corrected. AntibodiesI suggest the following topic sentence for this section.“Several antibodies were used to stain cells for cytokines and to measure polypeptides in RBC extracts by western blotting and they are briefly described below and their Research Resource Identifiers (RRIDs) given.”Author´s response: We have included this sentence. Western blotno suggestions Intracellular immunofluorescence staining and flow cytometry no suggestions Antiviral activity of conditioned mediumWould not the conditioned medium (CM) have virus that had not bound to RBC and thus not pelleted at 1600 rpm and so was in the CM? Please briefly discuss.Author´s response: The supernatants of RBCs exposed to IPNV were titrated and resulted in approximately 10 TCID50/mL or less. We have added the following sentence to clarify this item: “IPNV titer in the supernatants of IPNV-exposed RBCs resulted in 10 TCID50/mL or less, therefore viral presence in the supernatants was obviated”. Is ‘staining’ rather than “stain” meant in the following sentence and the next heading?“Finally, intracellular stain of IPNV foci was carried out.”Author´s response: It has been corrected. Intracellular stain of IPNV fociAdd to the end of the 3rd sentence “for 1 hour with the cells”.Author´s response: It has been corrected. MTT assaysAt the end of this section, a statement of how the absorbance readings are converted to percent viability should be given. I know that it is mentioned in the legend for Figure 3.Author´s response: The formula has been added.Software and statisticsI suggest the following modification to the 2nd sentence.“was used for preparing graphs and preforming statistical calculations”.Author´s response: It has been corrected. Ethics approvalno suggestions RESULTS IPNV did not infect trout RBCsI suggest the following recasting of the last sentence and addition of a new last sentence.“Moreover, flow cytometry analysis of control and IPNV-exposed RBCs for IPNV VP3 protein did not show significant differences (Figure 1C and D). Therefore, IPNV did not infect trout RBCs.”Author´s response: We have recasted the sentence as advised. IPNV exposure increased the expression of interferon-related antiviral immune genes and proteins in RBCsThe first sentence should be shortened as follows“to determine if IPNV would induce an antiviral response in RBCs.”Are there antiviral genes that are not immune genes?In other words, can the description be simplified to just ‘antivrial genes”?Author´s response: We have shortened the sentence and the description simplified to antiviral genes DISCUSSION2nd paragraphThe last two sentences could refer to the data of the ms for support.Author´s response: We have referred this affirmation to Figure 1.4th paragraphI wonder if the authors could briefly discuss the apparent constitutive expression of Mx in RBCs as seen in Panel D of Figure 2. Also could Mx be released from RBCs? In cattle Mx1 is found in exosomes (see Racicot K et al., 2012, Am J Reprod Immunol) and exosomes can be released by RBCs (see Danesh A et al., 2014, Blood).Author´s response: Yes, Figure S1 also highlights the elevated constitutive expression of Mx in RBCs, compared to other CHSE-214. Mx in RBCs exosomes is very interesting appreciation. However, Racicot et al (2012) concluded that “The results presented here show MX1 could play a role in the formation of vesicles secreted into the uterus and suggest involvement in basic cellular processes independent of its role during viral infections”. Therefore, although it is an interesting topic of investigation we would rather not to mention the possible link between Mx and RBCs exosomes in this manuscript since it is unknown whether it could be related to Mx antiviral function.FIGURES AND LEGENDS Figure 1In panel A write out hpe on the X axis.Author´s response: It has been writtenFigure 2Is the staining in the control of interest or just background? I mentioned this earlier under the Discussion. Could any of the cells in panel D be thrombocytes?Author´s response: Yes, we have discussed about it in the discussion section. In relation to panel D, none of those cells are thrombocytes. Figure 3Could FFU be written out here in the legend so figure stands alone? The legend mentions doing a two-way ANOVA but no mention of a post test is given, although the 1/5 supernatant dilution is identified with an asterisk?Author´s response: Yes, FFU/mL has been deleted from panel A, x axis, as it is indicated in the figure legend. On the other hand, Sidak´s multiple comparison test was performed and has been added to the legend.Figure 4 Title legend has a spelling mistake and is a bit misleading because panel B is not covered by the current title. I suggest the following title.“Reduction in RBC cytokine levels by IPNV and a possible mechanism”The legend should give more information so the figure can better stand by itself.Write out MFI (Mean Fluorescence Intensity) and FC (Flow Cytometry).At two places in the legend, mention is made of the Mann-Whitney test but the outcome is not clearly stated.Author´s response: Title of Figure 4 has been changed as follows: IPNV-exposure decreased cytokine levels in rainbow trout RBCs, and MFI and FC explained. In relation to the statistical analysis, Mann-Whitney test was performed for Fig4A and 4b, however, statistically significant differences were not observed." } ] } ]	1	https://f1000research.com/articles/6-1968
https://f1000research.com/articles/6-1832/v1	13 Oct 17	{ "type": "Research Note", "title": "Evaluation of electrochemiluminescence immunoassays for immunosuppressive drugs on the Roche cobas e411 analyzer", "authors": [ "Angela W.S. Fung", "Michael J. Knauer", "Ivan M. Blasutig", "David A. Colantonio", "Vathany Kulasingam", "Angela W.S. Fung", "Michael J. Knauer", "Ivan M. Blasutig", "David A. Colantonio" ], "abstract": "Background: Therapeutic drug monitoring of immunosuppressant drugs are used to monitor drug efficacy and toxicity and to prevent organ transplantation rejection. This study evaluates the analytical performance of semi-automated electrochemiluminescence immunoassays (ECLIA) for cyclosporine (CSA), tacrolimus (TAC) and sirolimus (SRL) on the Roche cobas e 411 analyzer at a major transplant hospital to identify method suitability and limitations. Methods: Residual whole blood samples from patients undergoing immunosuppressant therapy were used for evaluation. Experiments included imprecision, linearity, functional sensitivity, method comparisons and lot-to-lot assessments. Results: Total imprecision ranged from 3.3 to 7.1% for CSA, 3.9 to 9.4% for TAC, and 4.6 to 8.2% for SRL. Linearity was verified from 30.0 to 960.9 μg/L for CSA, from 1.1 to 27.1 μg/L for TAC, and from 0.5 to 32.3 µg/L for SRL. The functional sensitivity met the manufacturer’s claims and was determined to be <6.5 μg/L for CSA, 1.1 μg/L for TAC, and <0.1 µg/L for SRL (CV≤20%). Deming regression analysis of method comparisons with the ARCHITECT immunoassay yielded slopes of 0.917 (95%CI: 0.885-0.949) and r of 0.985 for CSA, 0.938 (95%CI: 0.895-0.981) and r of 0.974 for TAC, and 0.842 (0.810-1.110) and r of 0.982 for SRL. Deming regression analysis of comparisons with the LC–MS/MS method yielded slopes of 1.331 (95%CI: 1.167-1.496) and r of 0.969 for CSA, 0.924 (95%CI: 0.843-1.005) and r of 0.984 for TAC, and 0.971 (95%CI: 0.913-1.030) and r of 0.993 for SRL. Conclusions: The cobas e 411 ECLIA for CSA, TAC, and SRL have acceptable precision, linearity, and functional sensitivity. The method comparisons correlated well with the ARCHITECT immunoassay and LC–MS/MS and is fit for therapeutic drug monitoring.", "keywords": [ "Cyclosporine", "tacrolimus", "sirolimus", "immunoassay", "ECLIA" ], "content": "Introduction\n\nImmunosuppressive drugs (ISD), such as the calcineurin inhibitors (cyclosporine (CSA) and tacrolimus (TAC)) and mammalian target of rapamycin (mTOR) inhibitors (sirolimus (SRL) and everolimus), are critical to the maintenance of solid organ transplantation1. CSA is a cyclic undecapeptide and TAC (also known as FK-506) is a macrolide lactone. CSA binds to cyclophilin A/B and inhibits calcineurin. TAC binds to FK506-binding protein 12 (FKBP-12) to form the calcineurin inhibitory complex. Inhibition of calcineurin, a serine/threonine phosphatase, leads to altered calcium-dependent signal transduction, and decreases T-cell activation and downregulates anti-inflammatory response-related genes2,3. SRL (also known as rapamycin) is a 31-membered macrolide antibiotic that binds to FKBP-12 and allosterically targets the mTOR pathway, inhibiting cell cycle progression, T-cell proliferation and differentiation2. SRL has structural similarities to TAC and competes with TAC for FKBP-12 binding1,2. All three ISDs are characterized by having variable absorption, poor bioavailability, strong affinity to blood proteins, leukocytes, and/or erythrocytes, and metabolism via cytochrome CYP3A4/5 and efflux transport by P-glycoprotein2.\n\nTherapeutic drug monitoring is a mainstay in immunosuppressant therapy. ISDs have narrow therapeutic ranges2, high interindividual variability in pharmacokinetics and pharmacogenetics4,5, susceptibility to food- and drug-drug interactions6, and adverse consequences if plasma drug levels are not maintained5,7. Similar toxic effects have been described for CSA and TAC, due to their overlapping mechanism of action, and includes nephrotoxicity, hypertension, and neurotoxicity2. TAC is a more potent calcineurin inhibitor than CSA, due to increased affinity for FKBP-12 and the advantage of decreased nephrotoxicity, risk of hyperlipidemia and hypertension1,2,8. TAC, however, is more likely to cause post-transplantation diabetes1,9,10. SRL does not cause renal toxicities; however, long-term SRL use can induce leukopenia, thrombocytopenia, and dyslipidemia11,12. The target therapeutic range for each ISD may vary depending on type of organ transplanted, time from transplantation, co-administered drugs, and method of analysis.\n\nRecently, semi-automated electrochemiluminescence immunoassays (ECLIA) for the quantification of CSA, TAC, and SRL in whole blood were developed and made available by Roche Diagnostics (GmbH, Mannheim, Germany)13,14. In this study, we evaluated the analytical performance of ECLIA method for CSA, TAC, and SRL on the Roche cobas e411 analyzer and compared to the commonly used chemiluminescent microparticle immunoassay (CMIA) method on the Abbott ARCHITECT i2000 analyzer (Abbott Laboratories, Abbott Park, IL, USA). This is the first report on the evaluation of ECLIA SRL, and compares the performance of all three ISDs together.\n\n\nMethods\n\nEthical approval for this study was waived by the Research Ethics Board at the University Health Network in Toronto, Ontario, Canada (16-6312) for use of routine collected specimens for the evaluation of method performance. Residual EDTA whole blood specimens from 300 patients undergoing immunosuppressant therapy (either cyclosporine, tacrolimus, or sirolimus) at the University Health Network, and CAP proficiency testing samples were used in this evaluation. Samples were collected and either analyzed within the same day or stored as per manufacturer recommendations. Samples were thawed and equilibrated to room temperature for 30 minutes and mixed well prior to analysis. In accordance with stability studies on whole blood ISD specimens, samples were analyzed within three months of collection and did not undergo more than two freeze-thaw cycles15–18.\n\nThe cobas ECLIAs (Roche Diagnostics GmbH, Mannheim, Germany) for CSA, TAC, and SRL are based on the competition of analyte in sample with a ruthenium-labeled analogue. A voltage is applied and electrochemiluminescence signal is detected. Testing was performed according to the manufacturer’s instructions. Briefly, the samples (calibrators, QC, whole blood samples) were manually pretreated by combining 300 µL of whole blood with 300 µL of Universal ISD Sample Pretreatment Reagent (containing zinc sulfate and methanol) and vortexed for 10 seconds to lyse the red blood cells, precipitate proteins and extract the analyte. The samples were centrifuged for 4 minutes at 15,000 x g, and the supernatant was decanted for analysis. Analysis was performed within 30 minutes of preparation to prevent evaporation of the extracted samples. The ECLIA assays were calibrated five times during the evaluation by a 2-point calibration using calibrators traceable to pure standard materials reconstituted in whole blood matrix by gravimetrical methods.\n\nThe ARCHITECT CMIAs (Abbott Laboratories, Abbott Park, IL, USA) for CSA, TAC, and SRL are based on competition of analyte in sample with acridinium-labeled analogue. The samples were manually pretreated according to the manufacturer’s instructions and site-specific standard operating procedures. For CSA, 100 µL of Cyclosporine Solubilizing Reagent (4% saponin) and 400 µL of Cyclosporine Precipitation Reagent (zinc sulfate in methanol and ethylene glycol) was added to 200 µL of sample19,20. For TAC, 200 µL of sample was mixed with 200 µL of Tacrolimus Precipitation Reagent (zinc sulfate in methanol)21,22. For SRL, 150 µL of sample was mixed with 300 µL of Sirolimus Precipitation Reagent (zinc sulfate in >50% v/v DMSO and ethylene glycol), vortexed and heated at 42oC for 10 minutes23,24. All ISD samples were then vortexed for 10 seconds and centrifuged for 4 minutes at 15,000 x g. The supernatants were decanted into labelled tubes and assayed within 30 minutes of sample preparation. The ARCHITECT CMIAs are calibrated according to the site-specific standard operation procedures, with a 6-point 4-parameter logistic curve fit (4PLC, y-weighted) that is traceable to pure standard materials in a whole blood matrix by gravimetrical methods. Internal QC was evaluated with Bio-Rad Lyphochek Whole Blood ISD Controls levels 1, 3, and 4.\n\nThree levels of manufacturer multi-analyte QC materials (Roche Diagnostics PreciControl ISD levels 1, 2, and 3) and third-party multi-analyte QC materials (Bio-Rad Lyphochek Whole Blood ISD Controls levels 1, 3, and 4) were analyzed. QC samples were prepared and measured in duplicate, one run per day over 10 days. The acceptance criterion for total imprecision was based on the recommendation of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology (IATDMCT) expert consensus group of ≤ 10%25.\n\nResidual patient sample with levels 2–3 times the claimed limit of quantification (LoQ) was used to generate a series of dilutions with blank whole blood. The neat sample and dilutions were measured in triplicates within one day. The precision profile curve was used to calculate the LoQ concentration corresponding to a CV of 20% with the upper 95% confidence limit.\n\nSince there is a lack of elevated CSA and TAC patient specimen, CSA and TAC linearity were assessed using CAP EQA linearity materials (6 concentrations measured in duplicate). SRL linearity was assessed using a patient sample above the upper measuring range diluted with blank whole blood to 6 concentrations and measured in duplicate. The acceptance criterion was defined as slope of 1.00 ± 0.05 and deviation <10%.\n\nMethod comparison experiments were assessed where anonymized residual patient samples spanning the analytical measuring range for each analyte were measured one replicate per method. CSA samples concentrations ranged from 41.0 to 1808.0 μg/L, TAC ranged from 2.1 to 30.0 μg/L, and SRL ranged from 1.8 to 34.6 μg/L as determined by ARCHITECT CMIA. Roche ECLIA measurements were compared to ARCHITECT CMIA (n=100). To further elucidate the accuracy between immunoassays, a subset of samples was also analyzed by LC-MS/MS (n=20) (Supplementary File 1). Lot-to-lot assessment was also performed between two lots of reagents for each ISD (n=20). The slope, intercept, correlation coefficient r were calculated by Deming regression analysis. The acceptance criteria for method comparison were defined as a slope of 1.00 ± 0.15 and r of ≥ 0.95, meanwhile for lot-to-lot comparison were defined as a slope of 1.00 ± 0.05 and r of ≥ 0.95.\n\nMicrosoft Excel (version 1708, Microsoft Office) and/or EP Evaluator (version 7.0.0.307, Data Innovations) were used for statistical analysis.\n\n\nResults and discussion\n\nTo assess imprecision, three levels of manufacturer (Roche PreciControl) and third-party (Bio-Rad Lyphochek) multi-analyte QC materials were analyzed using one lot of reagents in duplicate, one run per day over 10 days (Table 1). For the PreciControl, the total imprecision was <7.1% for CSA, <9.4% for TAC, and <5.6% for SRL. Imprecision for CSA and TAC were comparable to other studies13,14. Our study additionally evaluated third-party QC performance on ECLIA ISD assays, a total imprecision of <4.7% for CSA, <6.3% for TAC, and <8.2% for SRL were determined. The imprecision goal of ≤10%, based on the recommendation of the IATDMCT expert consensus group, was achieved for all QC samples25. Overall, the ECLIA methods demonstrate acceptable precision.\n\n*Conventional unit: 1 µg/L = 1 ng/mL\n\nThe ECLIA methods offer a wider linear analytical measuring range for CSA and TAC than CMIA methods. ECLIA CSA, TAC, and SRL were linear up to 960.9 μg/L, 27.1 μg/L, and 32.3 μg/L, respectively. The higher upper limit allows TDM and pharmacokinetic analysis of ISD at different time points and peak concentrations offering additional flexibility26,27.\n\nThe claimed functional sensitivity of the ECLIA ISD methods are improved for TAC and SRL compared to CMIA ISD methods. The functional sensitivities were assessed and the precision profile was used to calculate the LoQ corresponding to a CV of 20% with upper 95% confidence limit. The functional sensitivities were determined to be <6.5 μg/L for CSA, 1.1 μg/L for TAC, and <0.1 μg/L for SRL, which meets the 2007 European consensus guideline and IATDMCT expert consensus group recommended LoQ of 20.0 μg/L for CSA and a LoQ of 1.0 μg/L for both TAC and SRL25,28.\n\nFor method comparison, anonymized residual patient samples spanning the analytical measuring range for each analyte were measured. CSA samples concentrations ranged from 41.0 to 1808.0 μg/L, TAC ranged from 2.1 to 30.0 μg/L, and SRL ranged from 1.8 to 34.6 μg/L as determined by CMIA. ECLIA ISDs measurements were compared to CMIA ISDs (n=100). The acceptance criteria were defined as a slope of 1.00 ± 0.15 and r of ≥ 0.95. Figure 1 shows the Deming regression and Bland-Altman analysis for CSA, TAC, and SRL. ECLIA and CMIA CSA (Figure 1A) showed good agreement with a slope of 0.917 (95% CI: 0.885-0.949), intercept of -15.2 (95% CI: -39.4-9.0), and r of 0.985. For TAC, the ECLIA TAC also showed good agreement with CMIA TAC (Figure 1B) with a slope of 0.938 (95% CI: 0.895-0.981), intercept of 0.2 (95% CI -0.4-0.8), and r of 0.974. Similar trends were observed by others (slopes of 0.87 for CSA, and 0.96-0.98 for TAC)14,29. Reported for the first time, method comparison of ECLIA and CMIA SRL (Figure 1C) showed a slope of 0.842 (95% CI: 0.810-1.110), intercept of 0.9 (95% CI: 0.4-1.4), and r of 0.982. Overall, all three ECLIA ISDs met our acceptance criteria, with SRL slightly exceeding the limit for the slope.\n\nMethod comparisons and Bland-Altman plots for (A) cyclosporine (CSA), (B) tacrolimus (TAC), and (C) sirolimus (SRL) between cobas e411 ECLIA and ARCHITECT i2000 CMIA.\n\nTo further examine ECLIA performance with CMIA, a subset of samples was also analyzed by LC-MS/MS (n=20). For both CSA immunoassays, a positive bias was observed when compared with LC-MS/MS, similarly observed by others13,29,30 (Figure S1). For TAC, both the ECLIA and CMIA TAC had good agreement with LC-MS/MS, also similarly observed by others for different cohorts of solid organ transplant14,21,29 (Figure S2). Reported for the first time, the ECLIA SRL compared to LC-MS/MS showed a slope of 0.971 (95% CI: 0.913-1.030), intercept of 2.4 (95% CI: 1.6-3.3), and r of 0.993 (Figure 2). Meanwhile, CMIA SRL compared to LC-MS/MS showed a slope 1.119 (95% CI: 1.051-1.187), intercept of 1.4 (95% CI: 0.5-2.4), and r of 0.993. Based on our small sample size, both ECLIA and CMIA SRL generally showed good correlation with LC-MS/MS, with ECLIA SRL with better agreement to LC-MS/MS.\n\nFor lot-to-lot comparisons, Deming regression analysis of 20 residual patient samples tested by 2 lots of reagent for CSA shows a slope of 0.998 (95% CI: 0.967-1.029), intercept of 8.5 (95% CI: -10.2-27.2), and r of 0.998. For TAC, a slope of 0.972 (95% CI: 0.936-1.008), intercept of -0.4 (95% CI: -0.8-0.0), and r of 0.997. And for SRL, a slope of 0.913 (95% CI: 0.841-0.985), intercept of 0.1 (95% CI: -0.9-1.2), and r of 0.988. All three ISDs had good correlation between 2 different lots of reagents.\n\nEvaluation on practical considerations included ease-of-use, throughput, and workflow of the method. The sample pretreatment for the ECLIA method is faster, simpler, and more convenient than CMIA method due to the use of a single universal sample pretreatment reagent and protocol for all three ISDs. Additionally, there is no heating step for the SRL ECLIA method, which leads to a simpler workflow. The ECLIA universal sample pretreatment reagent and protocol would enable better workflow, simpler sample handling and inventory control.\n\n\nConclusion\n\nIn conclusion, the overall analytical evaluation of the ECLIA method for CSA, TAC, and SRL met acceptable performance. ECLIA CSA showed better precision than our current CMIA CSA. ECLIA CSA and TAC showed better linearity range, and ECLIA TAC and SRL showed good functional sensitivity than CMIA methods. Method comparisons showed good correlations and agreement between ECLIA ISDs and CMIA ISDs.\n\n\nData availability\n\nDataset 1: File containing the raw data and a table of contents for the data file. doi, 10.5256/f1000research.12775.d18003331", "appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe thank the technologists and support staff at University Health Network and the Hospital for Sick Children in Toronto, Ontario, Canada for their contributions to this study. The cobas ECLIA ISD reagents used in the method evaluation were provided by Roche Diagnostics.\n\nRoche Diagnostics had no role in the design, analysis, or interpretation of this study.\n\n\nSupplementary material\n\nSupplementary File 1: Supplementary Methods.\n\nClick here to access the data.\n\nFigure S1: Method comparisons and Bland-Altman plots for cyclosporine (CSA) between (A) cobas e 411 ECLIA and LC-MS/MS, and (B) ARCHITECT i2000 CMIA and LC-MS/MS.\n\nClick here to access the data.\n\nFigure S2: Method comparisons and Bland-Altman plots for tacrolimus (TAC) between (A) cobas e 411 ECLIA and LC-MS/MS, and (B) ARCHITECT i2000 CMIA and LC-MS/MS.\n\nClick here to access the data.\n\n\nReferences\n\nHalloran PF: Immunosuppressive drugs for kidney transplantation. N Engl J Med. 2004; 351(26): 2715–29. PubMed Abstract \| Publisher Full Text\n\nMika A, Stepnowski P: Current methods of the analysis of immunosuppressive agents in clinical materials: A review. J Pharm Biomed Anal. 2016; 127: 207–31. PubMed Abstract \| Publisher Full Text\n\nOetjen E, Thoms KM, Laufer Y, et al.: The immunosuppressive drugs cyclosporin A and tacrolimus inhibit membrane depolarization-induced CREB transcriptional activity at the coactivator level. Br J Pharmacol. 2005; 144(7): 982–93. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKurzawski M, Droździk M: Pharmacogenetics in solid organ transplantation: genes involved in mechanism of action and pharmacokinetics of immunosuppressive drugs. Pharmacogenomics. 2013; 14(9): 1099–118. PubMed Abstract \| Publisher Full Text\n\nMillner L, Rodriguez C, Jortani SA: A clinical approach to solving discrepancies in therapeutic drug monitoring results for patients on sirolimus or tacrolimus: Towards personalized medicine, immunosuppression and pharmacogenomics. Clin Chim Acta. 2015; 450: 15–8. PubMed Abstract \| Publisher Full Text\n\nAllison TL: Immunosuppressive Therapy in Transplantation. Nurs Clin North Am. 2016; 51(1): 107–20. PubMed Abstract \| Publisher Full Text\n\nAgrawal YP, Cid M, Westgard S, et al.: Transplant patient classification and tacrolimus assays: more evidence of the need for assay standardization. Ther Drug Monit. 2014; 36(6): 706–9. PubMed Abstract \| Publisher Full Text\n\nPham SM, Kormos RL, Hattler BG, et al.: A prospective trial of tacrolimus (FK 506) in clinical heart transplantation: intermediate-term results. J Thorac Cardiovasc Surg. 1996; 111(4): 764–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoore R: New-onset diabetes after renal transplantation: comparing ciclosporin and tacrolimus. Nat Clin Pract Nephrol. 2008; 4(1): 20–1. PubMed Abstract \| Publisher Full Text\n\nVincenti F, Friman S, Scheuermann E, et al.: Results of an international, randomized trial comparing glucose metabolism disorders and outcome with cyclosporine versus tacrolimus. Am J Transplant. 2007; 7(6): 1506–14. PubMed Abstract \| Publisher Full Text\n\nHalleck F, Duerr M, Waiser J, et al.: An evaluation of sirolimus in renal transplantation. Expert Opin Drug Metab Toxicol. 2012; 8(10): 1337–56. PubMed Abstract \| Publisher Full Text\n\nGummert JF, Ikonen T, Morris RE: Newer immunosuppressive drugs: a review. J Am Soc Nephrol. 1999; 10(6): 1366–80. PubMed Abstract\n\nVogeser M, Shipkova M, Rigo-Bonnin R, et al.: Multicenter analytical evaluation of the automated electrochemiluminescence immunoassay for cyclosporine. Ther Drug Monit. 2014; 36(5): 640–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShipkova M, Vogeser M, Ramos PA, et al.: Multi-center analytical evaluation of a novel automated tacrolimus immunoassay. Clin Biochem. 2014; 47(12): 1069–77. PubMed Abstract \| Publisher Full Text\n\nHolt DW, Lee T, Johnston A: Measurement of sirolimus in whole blood using high-performance liquid chromatography with ultraviolet detection. Clin Ther. 2000; 22(Suppl B): B38–48. PubMed Abstract \| Publisher Full Text\n\nCapone D, Gentile A, Polichetti G, et al.: Stability of sirolimus and everolimus measured by immunoassay techniques in whole blood samples from kidney transplant patients. Int J Immunopathol Pharmacol. 2008; 21(2): 297–307. PubMed Abstract \| Publisher Full Text\n\nDubbelboer IR, Pohanka A, Said R, et al.: Quantification of tacrolimus and three demethylated metabolites in human whole blood using LC-ESI-MS/MS. Ther Drug Monit. 2012; 34(2): 134–42. PubMed Abstract \| Publisher Full Text\n\nAnnesley TM, Hunter BC, Fidler DR, et al.: Stability of tacrolimus (FK 506) and cyclosporin G in whole blood. Ther Drug Monit. 1995; 17(4): 361–5. PubMed Abstract\n\nBrate EM, Finley DM, Grote J, et al.: Development of an Abbott ARCHITECT cyclosporine immunoassay without metabolite cross-reactivity. Clin Biochem. 2010; 43(13–14): 1152–7. PubMed Abstract \| Publisher Full Text\n\nCyclosporine for Abbott Architect iSystems [package insert]. Abbott Laboratories, Abbott Park, IL USA, 2008. Reference Source\n\nBazin C, Guinedor A, Barau C, et al.: Evaluation of the Architect tacrolimus assay in kidney, liver, and heart transplant recipients. J Pharm Biomed Anal. 2010; 53(4): 997–1002. PubMed Abstract \| Publisher Full Text\n\nTacrolimus for Abbott Architect iSystems [package insert]. Abbott Laboratories, Abbott Park, IL USA, 2009. Reference Source\n\nSchmid RW, Lotz J, Schweigert R, et al.: Multi-site analytical evaluation of a chemiluminescent magnetic microparticle immunoassay (CMIA) for sirolimus on the Abbott ARCHITECT analyzer. Clin Biochem. 2009; 42(15): 1543–8. PubMed Abstract \| Publisher Full Text\n\nSirolimus for Abbott Architect iSystems [package insert]. Abbott Laboratories, Abbott Park, IL USA, 2009. Reference Source\n\nSeger C, Shipkova M, Christians U, et al.: Assuring the Proper Analytical Performance of Measurement Procedures for Immunosuppressive Drug Concentrations in Clinical Practice: Recommendations of the International Association of Therapeutic Drug Monitoring and Clinical Toxicology Immunosuppressive Drug Scientific Committee. Ther Drug Monit. 2016; 38(2): 170–89. PubMed Abstract \| Publisher Full Text\n\nOellerich M, Armstrong VW: Two-hour cyclosporine concentration determination: an appropriate tool to monitor neoral therapy? Ther Drug Monit. 2002; 24(1): 40–6. PubMed Abstract \| Publisher Full Text\n\nKnight SR, Morris PJ: The clinical benefits of cyclosporine C2-level monitoring: a systematic review. Transplantation. 2007; 83(12): 1525–35. PubMed Abstract \| Publisher Full Text\n\nWallemacq P, Armstrong VW, Brunet M, et al.: Opportunities to optimize tacrolimus therapy in solid organ transplantation: report of the European consensus conference. Ther Drug Monit. 2009; 31(2): 139–52. PubMed Abstract \| Publisher Full Text\n\nToole B, Gechtman C, Dreier J, et al.: Evaluation of the New Cyclosporine and Tacrolimus Automated Electrochemiluminescence Immunoassays under Field Conditions. Clin Lab. 2015; 61(9): 1303–15. PubMed Abstract \| Publisher Full Text\n\nWallemacq P, Maine GT, Berg K, et al.: Multisite analytical evaluation of the Abbott ARCHITECT cyclosporine assay. Ther Drug Monit. 2010; 32(2): 145–51. PubMed Abstract \| Free Full Text\n\nFung AWS, Knauer MJ, Blasutig IM, et al.: Dataset 1 in: Evaluation of electrochemiluminescence immunoassays for immunosuppressive drugs on the Roche cobas e411 analyzer. F1000Research. 2017. Data Source" }	[ { "id": "26931", "date": "20 Oct 2017", "name": "Jennifer Shea", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFung et al. present data evaluating three new ECLIA assays for the immunosuppressant drugs cyclosporine, tacrolimus, and sirolimus on a Roche Cobas e411 analyzer. Overall the study design is sound generally following CLSI guidelines for method validation. The introduction provides a thorough background on the drugs/assays being evaluated and the methods section clearly describes each portion of the method validation that would easily allow another researcher to replicate the study if desired. The results/discussion section succinctly presents their data in a cohesive and systematic manner and the authors do a great job at highlighting the novel aspects of their study. Their conclusion that these three new assays are fit for use for therapeutic drug monitoring is supported by the data presented.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "26927", "date": "30 Nov 2017", "name": "Julie Shaw", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere the authors describe evaluation of three immunosuppressant drug immunoassays on the Roche Cobas e411 analzyer. The authors evaluated the performance of assays for cyclosporine, tacrolimus and sirolimus on the system, which included imprecision, linearity and functional sensitivity studies as well as method comparisons and lot-to-lot comparisons. They compared the assay performance to the performance of the same assays on the Abbott immunoassay system and to LC MS/MS assays for the same analytes.\n\nI have a few comments that, if the authors could address, I feel would strengthen the manuscript.\n\nIn the methods section, it’s not clear to me whether the measurements were performed using the Roche, Abbott and LC-MS/MS methods on the same day. It is mentioned that some specimens were stored prior to analysis. Could the authors clarify this?\n\nWhat are the immunoassays and LC-MS/MS methods traceable to? Is there a standard? This information would be helpful.\n\nThe authors mention that the Roche assays were calibrated five times over the course of the study. Is this in fitting with the manufacturer’s recommendations? How does this calibration frequency compare to the Abbott system?\n\nThe authors should discuss the limitation of performing imprecision studies using only one lot number of reagent.\n\nThe authors should include a description of the LC MS/MS method that was used. There is currently no mention of this methodology until the results section.\n\nThere are several grammatical errors throughout the manuscript and I would recommend that the authors read it through carefully to correct these.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-1832
https://f1000research.com/articles/6-1083/v1	07 Jul 17	{ "type": "Review", "title": "A sequencer coming of age: De novo genome assembly using MinION reads", "authors": [ "Carlos de Lannoy", "Dick de Ridder", "Judith Risse", "Dick de Ridder", "Judith Risse" ], "abstract": "Nanopore technology provides a novel approach to DNA sequencing that yields long, label-free reads of constant quality. The first commercial implementation of this approach, the MinION, has shown promise in various sequencing applications. This review gives an up-to-date overview of the MinION's utility as a de novo sequencing device. It is argued that the MinION may allow for portable and affordable de novo sequencing of even complex genomes in the near future, despite the currently error-prone nature of its reads. Through continuous updates to the MinION hardware and the development of new assembly pipelines, both sequencing accuracy and assembly quality have already risen rapidly. However, this fast pace of development has also lead to a lack of oversight in the expanding landscape of analysis tools, as performance evaluations are outdated quickly. Now that the MinION is approaching a state of maturity, a thorough comparative benchmarking effort of de novo assembly pipelines may be at place. An earlier version of this article can be found on BioRxiv.", "keywords": [ "nanopore sequencing", "MinION", "\\textit{de novo} sequencing", "review" ], "content": "Introduction\n\nThe development of novel genome sequencing methods has been a major driving force behind the rapid advancements in genomics of the last decades. Notably, the advent of second generation sequencing (SGS) provided researchers with the required throughput and cost-efficiency to sequence many more genomes than was previously deemed feasible. Recent years saw the dawn of what can be perceived as a third generation; one that allows amplification-free reading of single DNA molecules in long consecutive stretches [van Dijk et al., 20141, review.] Currently, this new generation is dominated by two methods: nanopore sequencing and single-molecule real time (SMRT) sequencing, championed by Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio), respectively.\n\nConceptually, nanopore sequencing is easier to explain than most other sequencing methods. An electrical potential is applied across an insulating membrane in which a single small pore is inserted. A DNA strand is pulled through the pore and the sequence is inferred from the characteristic way in which the passing base combinations influence the current. In 1989, David Deamer roughly sketched this concept as it is applied today, although it took more than two decades of key innovations to bring the concept to fruition2. Since the introduction of the first commercially available nanopore sequencing device, ONT’s MinION, and the start of the MinION Access program (MAP) in 2014, the field of nanopore sequencing has been advancing at a rapid pace; both new applications and improvements to existing ones are published on a regular basis.\n\nThe advantages of the MinION over other sequencing devices are numerous. Both its size, roughly that of a cellphone, and its cost, a thousand dollars for a starter kit, are a mere fraction of that of competitors. Running the MinION is also reasonably time- and cost-effective; an 48-hour sequencing run currently costs about 800 dollarsi and typically yields about 5 Gbase3, although recently yields as high as 15.7 Gbase have also been reported (Baptiste Mayjonade, personal communication July 6th, 2017). Furthermore, the technique does not rely on any labeling techniques to recognize different bases, while Sanger, SGS methods and SMRT do require some form of labeling of nucleotides. Amplification by PCR is optional for the MinION, while this step is mandatory for Sanger and SGS-methods. Not only does omitting these steps simplify sample preparation for MinION samples, it also helps to avoid errors and biases (e.g. the CG-bias for PCR) and allows detection of modified bases4. Finally, the maximum read length produced by the MinION is many times greater than that of both second-generation and Sanger sequencing and only paralleled by SMRT sequencing, which is highly advantageous in resolving repeat sequences.\n\nThe most prominent disadvantages of the MinION, with respect to its competitors, are the lower signal-to-noise ratio, stochasticity introduced by its biological components, and the resulting high error rate of basecalling. Indeed, the MinION is a product in development and the used materials (i.e. membranes, nanopores and buffers) are still being optimized. Furthermore, it is thought that significant improvements are still possible in the software pipelines that translate current signal to DNA sequence, even though recent efforts have increased the base-calling accuracy dramatically, from around 85% in the first half of 20165–7 to over 95% in March of 2017.\n\nIn this review, an up-to-date overview of several aspects of nanopore sequencing is provided. First, the physical sequencing process as it takes place inside the MinION is outlined. Then, the general structure of analysis pipelines is described, along with currently available software implemented in these pipelines and their respective strengths and weaknesses. It should be noted that nanopore sequencing is a rapidly advancing field. While some work discussed in this chapter is considered cutting-edge at the moment of writing, the reader is advised to keep the publication date of said work in mind.\n\n\n1 Physical basis of DNA sequencing using nanopores\n\nIn a nutshell, the underlying principle of nanopore sequencing can be explained as follows: a microscopic opening wide enough to allow single-stranded DNA to pass - the nanopore - is introduced in an insulating membrane between two compartments filled with saline solution and an electric potential is applied across it. DNA strands are then added to one compartment and allowed to diffuse toward the nanopore, where they are captured by the electric field and threaded through the pore. While a strand is passed through, the characteristic way in which the bases influence the electric current through the nanopore is measured. These measurements can then be decoded to retrieve the sequence of the DNA strand (Figure 1).\n\nFrom left to right, double-stranded DNA with attached motor protein attaches to a pore protein in an insulating membrane. The applied potential pulls one strand through the pore, while the motor protein unzips the DNA in a step-wise fashion. After the DNA has been unzipped completely and one strand has passed through, the complex detaches from the pore entrance and the pore is ready to receive another strand. Image courtesy of Oxford Nanopore Technologies Ltd5.\n\nIn recent years, several key discoveries have brought reliable nanopore sequencing closer at a rapid pace. In a step-by-step exploration of the sequencing process, these discoveries will be discussed next.\n\nChoice of pore: Biological versus solid-state Nanopore sequencing efforts are sub-categorized in two groups based on the choice of nanopore. Most current efforts implement biological nanopores, which are protein polymers derived from naturally occurring counterparts. Through genetic engineering, biological nanopores are modifiable in terms of dimensions and placement of electrical charge. These properties are also highly reproducible from one pore to the next. Functionality can be further modified by attaching compatible enzymes to the pore opening. Like their naturally occurring counterparts however, they need to be embedded in lipid bilayers, which are generally prone to disruption, particularly when exposed to varying electrical potentials. In the MinION, this was partly solved by constructing membranes out of a more stable single layer of polymers, rather than the traditional bilayer. On the other hand, solid-state nanopores are made by burning openings in a synthetic membrane using a focused electron or ion beam8. Contrary to biological nanopores, solid-state nanopores are compatible with a wide range of strong and chemically stable materials with equally diverse properties. Pores are also more easily parallelized and integrated in electrical readout circuits. A major disadvantage at the moment is the reproducibility of the pore dimensions. They also do not combine as easily with modifying enzymes. As a result, solid-state nanopores currently produce noisier and less easily interpretable signals than biological nanopores. In the following, the focus will lie on biological nanopore sequencing and the term nanopore will refer to the biological kind.\n\nStructure and charge of the nanopore One important structural property that makes a biological pore suitable for DNA sequencing is a constriction site at which the passing strand exerts the most influence on the electrical current. The length of the constricting passage largely determines how many bases simultaneously influence the electrical current and thus the number of bases k that is “read” simultaneously at a given time. k should be kept low enough to allow recognition of a signature current for each different combination of k bases, or k-mer, and high enough to allow for some overlap between subsequent k-mers, as this benefits basecalling accuracy by allowing every base to be read k times. Modified versions of both pore proteins that have seen application in the MinION, MspA (designated R7 by ONT) and the currently used CsgG9 (designated R9, Figure 2), which have a constricted passage that allows detection of manageable k-mer lengths. For the 10Å-long constriction of the CsgG pore, basecalling models assume that five nucleotides influence the current at one given time sufficiently to discern all different nucleotide combinations, and thus 5-mers are called.\n\nPositive and negative residues are colored blue and red, respectively. Image generated by the authors using PyMOL v1.7.0.0. PDB ID: 4UV39.\n\nFor sequencing to commence, a DNA strand first needs to diffuse towards one side of the pore, referred to as the cis-side, where it is captured by the electric field resulting from the applied potential. It is then threaded through the pore and extruded at the other end, called the trans-side.\n\nTwo forces should be considered. First and most importantly, the electrophoretic force induced by a positive electric potential applied at the trans-side attracts the negatively charged DNA and pulls it in. As negative particles leave the cis-side and positive particles simultaneously move in the opposite direction, a positively charged zone forms around the cis entrance of the pore, strengthening attraction of DNA strands. Secondly, strand translocation is influenced by the electro-osmotic flow (EOF), the force induced by the net water and ion flow through the pore. While a DNA strand is in the pore, the EOF normally opposes the direction of the electrophoretic force and thus of translocation; however this effect is relatively minor.\n\nThrough iterative optimization of internal architecture, it was found that positive internal surface charges are important for efficient DNA capture10,11, while base recognition was found to improve with bulky or hydrophobic amino acid side chains placed at the constriction site, as these direct ion flow toward the DNA strand12. Although the structure of the modified CsgG pore9 implemented in the current MinION flow cells (R9-type) has not been publicly released by ONT, modifications to these properties have likely been made.\n\nProcessive control It should be noted that the processive speed of the strand is too high without any further modifications (between 2·106 and 10·106 bases/s in wild-type MspA)10. Currently, the most successful way to exert control over the speed has proven to be the addition of a motor protein, such as phi29 DNA polymerase (DNAP)13 or a helicase14. In a preparatory step, a poly-T or \"leader\" adapter is attached to the double-stranded DNA. The motor protein attaches to this adapter, but due to specialized bases in the adapter sequence (left unspecified by ONT), it cannot unzip it at this stage (see community technical document on this subject). Once the complex is adjacent to the cis-side of the pore, the leader adapter previously blocking the motor protein is released, presumably due to the force exerted on the strand as demonstrated by15 and described in Heron et al., 201616. The DNA is then fed through the pore by the motor protein, where it can now be \"read\" at a reasonably regular pace. Presumably, a modified helicase is currently used as motor protein in the MinION14. The latest release of this motor protein at the moment of writing (dubbed E8) maintains an average throughput speed of 450 bases/s.\n\nReading the DNA strand During a MinION sequencing run, the potential over the membrane is kept stable, while the electrical current (in the pA-range) is sampled at a frequency in the kHz range (Figure 3). This signal is characteristic for the subsequent bases moving through the pore and will ultimately serve as the basis for basecalling. As the amount of electrolyte is increasingly depleted during the run, the applied potential (typically starting at -180mV) is further decreased by 5 mV per two hours of runtime and increased by 5mV when the MinION switches to another set of wells filled with fresher buffer (see next section).\n\nThe data used in the construction of this figure is mock data.\n\nWhile the MinION can read the first strand of a dsDNA-stretch that is threaded through the pore - by definition, the template strand - and discard the complementary strand, it is possible to instead read the complementary strand immediately after the template, thus performing a second read of the same stretch (Figure 4). Combining reads of both strands has been shown to increase sequencing accuracy significantly5. So far, ONT has provided two ways of doing so; 2D-sequencing and its successor 1D2-sequencing (versus 1D sequencing if only the template strand is read). To enable 2D-reads, an abasic hairpin sequence (i.e. a bare backbone of DNA) is attached to the dsDNA, connecting the 3’-end of the template and the 5’-end of its complement. After the template strand has been read, the hairpin structure is threaded through the pore, recognizable due to its abasic nature, followed by the complement strand. The mirroring reads are then decoded jointly so that any sequencing errors may be corrected. It should be noted however, that decoding 2D-reads comes at a high computational cost. Furthermore, the exact start and end of the hairpin is not always clear from the signal. The hairpin may also ligate strands in other ways than the intended one; for example, multiple reads may be linked together by hairpins into chimeric reads17. Lastly, the read of the complement strand is often less accurate and threaded through the pore at lower speed5, reportedly due to structural changes in the strand caused by the template and complement rezipping while the complement strand is still passing through the pore. In May of 2017, 1D2-read chemistry was introduced as a replacement of 2D-read chemistry. 1D2 chemistry does not join the two strands, but rather attaches an adapter to the complement strand, allowing it to attach to the membrane while the template strand is read. Shortly after the template strand has completely left the pore, the complement strand is pulled in and sequenced. Without the hairpin connecting the strands, recognition of its start and end is no longer required and, as the strands are effectively read as two separate reads, no drop in read quality is observed for the complement strand.\n\n(A) When using 1D chemistry, only the template strand (blue) is threaded by the motor protein (green) and read. The complement strand (red) is discarded at the cis side of the pore. The tethers (dark-green) allow for selection of properly ligated complexes during sample preparation and attach to the membrane to increase the availability of strands near pores during sequencing. (B) The now-deprecated 2D chemistry connected template and complement strand using a hairpin, thus allowing sequencing of the complement strand immediately after the template strand. An additional tether that attached to the hairpin allowed for selection of correctly ligated strands during sample preparation. (C) 1D2 chemistry, the successor of 2D, also allows sequencing of both strands, but rather than attaching the two, the complement strand is tethered to the membrane while the template is sequenced. After the template strand is threaded through, the complement strand is drawn in and the tether is pulled loose. Based on Jain et al., 201518 by permission from Macmillan Publishers Ltd: Nature Methods, copyright(2015), the ONT kit content description, and ONT’s technical update of March 2017.\n\nChannel parallelization Lastly, throughput can be greatly increased by reading the signal from multiple pores in parallel. The current generation of the MinION’s disposable cartridges, called flow cells, can read the signal of up to 512 pores in parallel (Figure 5). The flow cell is equipped with 2048 wells, which are connected in groups of four to multiplexers (MUXs), the switches that control which of the four cells per group is controlled and read out by the circuits. During the initial platform quality check, DNA strands (of unreleased source and sequence), present in the buffer with which the flow cells are shipped, is sequenced to discern wells containing precisely one correctly inserted pore from wells in which correct pore insertion has failed (e.g. no or multiple pores were inserted; see ONT platform quality check protocol). The latter scenario may occur, as the insertion of pores is a stochastic process. In a second quality check, the MUX scan, each MUX chooses three wells in order of signal quality and begins readout in the best-quality well. As well quality is expected to decline during the run, the standard protocol switches to the second-best quality pore after eight hours, and the third-best quality after another eight hours. This way, the best and most output is expected in the first part of the run. While a run using a group of wells is in progress, the circuits connected to the MUXs regulate the current in each selected well individually. This also allows expelling of eventual blockades from a pore, by temporarily reversing the current in the affected well while the rest of the wells continue to function normally.\n\nLarge circles denote wells in the grid, small black circles denote inserted nanopores. Each group of four wells is controlled by a multiplexer (MUX). During an initial quality check, wells that are unusable due to erroneous pore insertion are marked unusable (red circles). Right before sequencing, the wells are tested a second time and three wells per MUX are ranked on signal quality (if possible). Sequencing of the sample will then commence, starting read-out from the best-performing well (green) and switching to second and third best (yellow) after eight hours each.\n\n\n2 Currently available software for MinION sequencing data\n\nFollowing the process in section 1, a current signal is obtained that needs to be translated into the corresponding DNA sequence. Pipelines used for this purpose generally subdivide the signal into discrete stretches corresponding to a particular set of k bases, called events, and then attempt to find the most likely set of bases for each event6,7. Ideally, the processive control exerted by the motor protein causes the strand to move one base further through the pore between each event transition, such that each base in the k-mer is read k times. Due to irregularity in the motor protein functioning and noise in the signal, this is not always the case. Three transition scenarios may be observed. (I) If the strand progresses exactly one base further from one event to the next, this is called a \"step\". Alternatively, (II) the next event may denote a k-mer located one or multiple bases further upstream, which is referred to as a \"skip\", or (III) the segmentation process may incorrectly split up an event in two events that correspond to the same k-mer, which is called a \"stay\". Steps, skips and stays may be identified by comparing the bases in the read k-mers; however, this proves more difficult in short tandem repeat regions, as subsequent events would look the same for multiple transition scenarios. More specifically, in a homopolymer stretch of length k or longer, the difference between skips, steps and stays may be unclear. Similarly, repeats of m bases may cause problems in discerning skips of (multiples of) m bases and stays. Such ambiguities have been shown to be a major cause of deletions in current basecallers6,18–20.\n\nTo assess the quality of basecalling performance, a 3.6 kbase calibration strand derived from the Lambda genome may be added to the sample. The software controlling the MinION (MinKNOW) automatically detects reads derived from the Lambda genome and separates those from the sample reads. Some software tools use these strands to parameterize basecall correction algorithms21.\n\nOnce reads have been basecalled, they may serve several purposes. If SGS reads are available, then those may then be mapped to the MinION reads to correct sequencing errors pre-assembly22, or to create large low-error contigs. The latter goal may be achieved either by using MinION reads as scaffolds to properly align short reads23,24 or by creating short accurate seed regions from short reads, the gaps between which are then bridged by MinION reads20. Both approaches were shown to result in accurate and highly continuous de novo assemblies and identification of repeats that were collapsed in SGS-only assemblies20,22. In MinION-only assembly pipelines, overlaps between the basecalled sequences are sought to create initial assemblies. Currently, the error-prone nature of MinION reads necessitates a post-assembly error correction (\"polishing\"), in which raw reads are mapped again to the assemblies until a better consensus is reached25. Below, the different tools MinION users have at their disposal to fill in the steps in this pipeline are discussed, along with their strengths and weaknesses.\n\nIt should be noted that the presented list of tools is not exhaustive; the focus of this section lies on tools that can be used in de novo MinION sequencing, without the need for other sequencing methods. Furthermore, only tools that provide a full solution to their respective step in the assembly pipeline are reported here. Notably, stand-alone read mapping tools exist, which may be used in combination with the assembly tools described in this section; however, detailed descriptions are omitted for the sake of brevity. Readers with an interest in these alternatives may want to look into GMAP26, Graphmap27 and MinHash alignment process (MHAP)28. As current assemblers either include their own error correction module29 or work with uncorrected reads20,30–32, stand-alone pre-assembly error correction tools are excluded as well.\n\nBefore basecalling takes place, current basecalling tools require the continuous signal to be divided into discrete sections, called events, each supposedly representing one combination of bases. Furthermore, the poly-T lead preceding the sequence needs to be removed, and, if 2D chemistry is used, the signal derived from the template strand needs to be separated from that of the hairpin and the complement strand. The latter process is commonly referred to as segmentation.\n\nFor locally basecalled reads, both segmentation and subsequent event detection are performed by the MinKNOW software provided by ONT. The software identifies the hairpin by a characteristic double current spike, while a long poly-T signal denotes the lead. For event detection, MinKNOW was reported to calculate a simple t-statistic between sliding adjacent windows of set size. Peaks in the t-statistic above a certain threshold are then assumed to signify the border between two segments. Optionally, the raw current signal can also be reported for further analysis.\n\nSeveral dedicated basecalling tools are already available to MinION users. In this section, the underlying principles and implementation of these tools are explored, along with their reported subsequent strengths and weaknesses. It should be noted that an all-inclusive fair comparison, in which all basecallers are optimized for the latest MinION chemistry and then run on the same dataset, is currently lacking. Any comparison in this chapter is therefore based on accuracy reports made by the authors of the open-source basecallers in their publications (i.e. between the Metrichor basecallers and their own).\n\nMetrichor basecallers Metrichor, a spin-off company of ONT and its main developer of proprietary analysis software, maintains a range of basecallers that have remained the go-to option for most MinION users. Initially, the Metrichor basecallers relied on hidden Markov models (HMM) to find the biological sequence underlying the segmented signal. As of early 2016, this model was replaced by a more accurate recurrent neural network (RNN)-implementation. Currently several RNN-based versions exist under different names; Albacore, Nanonet and the MinKNOW integrated basecaller. Albacore and the MinKNOW version are stable versions intended for regular MinION users. As of Albacore v1.0.1 and MinKNOW v1.6.11, both were reported to improve homopolymer calling through the implementation of a transducer, a feature that was transferred from the Scrappie basecaller (see below). Nanonet is an unsupported version under continuous development and makes use of the CURRENNT library to implement its RNN33. A cloud-based version was previously integrated in the EPI2ME platform, but this service has been discontinued. Due to the proprietary nature of the software, the source code of Albacore and the MinKNOW basecaller is currently only open to developer users, while that of Nanonet is openly available. The community manual describes the general procedure followed by all Metrichor basecallers as follows. Long reads are broken up into partially overlapping stretches, referred to as chunks. This keeps the Viterbi-decoding process, required after the RNN has determined k-mer probabilities, tractable. The RNN then assigns a likelihood for every possible five-mer to each event in the chunks.\n\nMetrichor basecallers were reported to use a bidirectional long-short term memory (BLSTM)-RNN. This architecture allows the RNN to overcome the vanishing gradient problem, encountered often during the training of deep neural networks, and the usage of multiple previous events in the sequenceii.\n\nThe RNN assigns probabilities for each k-mer to each event, which are used to estimate basecalling quality scores (i.e. q-scores). Five-mers that would entail a forbidden move, considering the likelihood of surrounding k-mers, are removed at this point. The maximum likelihood sequence is then extracted from the k-mer likelihoods through Viterbi decoding. Finally, basecalled chunks are merged again.\n\nThe accuracy of the Metrichor basecallers is currently considered to be the highest of all available basecallers. Before the switch to RNNs, an international performance analysis estimated the accuracy at 88% for 2D basecalling5. David et al. reported an accuracy of approximately 68%6 for 1D basecalling on human data, while Boza et al. reported 77% for 1D and 87% for 2D on Escherichia coli and Klebsiella pneumoniae genomes. After moving to an RNN-implementation (as well as numerous improvements in used chemistry and hardware) this percentage has risen to over 95% (see here for a publicly available dataset supporting this), even before the mentioned correction of homopolymer regions in Albacore and MinKNOW was applied.\n\nScrappie A basecaller by Metrichor and platform for ongoing development, Scrappie was the first basecaller reported to specifically address homopolymer basecalling, through the implementation of what is referred to as a transducer. It was written in C and its source code is publicly available. In previous MinION sequencing efforts, correctly determining the length of homopolymers proved challenging, as the separation between states in a homopolymer stretch was not clear20,21,35. Scrappie can process the raw current signal, rather than the event-detected data.\n\nAs an initial performance assessment, an early-development version of Scrappie was run in parallel to other Metrichor basecallers on reads of the human chromosome 20. It was found that Scrappie indeed called homopolymer stretches more accurately than the EPI2ME basecaller and Nanonet; homopolymeric stretches of up to 16 bases were called accurately (although a slight over estimation of repeat units was seen between 5 and 15 bases), after which the accuracy fell. In contrast, the EPI2ME basecaller and Nanonet failed to call stretches longer than five bases. The effect was also shown in an analysis of 5-mer counts; of the tested basecallers, Scrappie stayed closest to the reference genome and did not under-represent homopolymeric 5-mers3. Although these results are promising, a thorough assessment of Scrappie in a later stage of development is required to evaluate the true potential of the transducer-based homopolymer calling.\n\nNanocall Nanocall6 was the first open-source basecaller for the MinION offered as an alternative to the proprietary Metrichor software. It was written in C++. Nanocall accepts the segmented signal from MinKNOW and assigns k-mers to the events using a HMM.\n\nAn HMM assumes that, in a sequence of events, the probability of the next event being of a certain nature - its state - is dependent only on the current state. This makes sense for the basecalling of subsequent k-mers, as the prefix of the next k-mer that is called should be the suffix of the previous. The \"hidden\" property of HMMs refers to the fact that the sequence of states - the path - is not known. Instead, it has to be inferred with some probability from a derived signal. In the case of a basecaller, this signal consists of electrical current levels which are derived of a sequence of k-mers. The goal of the HMM is to find the path that is most likely to have emitted the given signal (for a more thorough introduction to HMMs, see 36).\n\nAs described by David et al.6, the HMM underlying Nanocall takes stay, step and skip scenarios into account at each event transition (Figure 6). Although skips of more than one base may occur in a sequence, Nanocall only considers one-base skips to cut down on computational requirements, thus reducing the number of neighboring states to 21 (i.e. stay, four different steps and sixteen different one-base skips). During an initial training phase, state transition probabilities for stays and skips of any number of bases, pstay and pskip respectively, are estimated using the Baum-Welch algorithm (pstep is derived as 1 − pstay − pskip)37. The transition probability for exactly one base pskip1 is then derived as pskip1 = pskip /(1 + pskip). Note that all sixteen one-base skips have the same transition probability (i.e. 116 · pskip1), as do the four step scenarios (14 · pstep). To calculate emission probabilities, Nanocall relies on ONT’s pore models, which provide parameters for current level distributions per k-mer, which can be fitted to individual reads. The most probable sequence is obtained through Viterbi decoding.\n\nThe path is chosen by maximizing the product of transition probabilities pstay, pstep and pskip1 (modified by fractions in the case of skips and steps, as there are multiple states to which these transitions could lead) and emission probabilities of k-mers. The data used in the construction of this figure is mock data.\n\nPerformance of Nanocall at the moment of its publication, in March of 2016, was reportedly on par with that of 1D analysis by the Metrichor basecallers, or around 68% accuracy6. However, unlike the Metrichor basecallers, Nanocall does not support 2D analysis, which allowed for a much higher accuracy in the comparison by David et al. (around 85%)6. Since the Metrichor basecallers switched to a recurrent neural network-approach, its 1D accuracy proved significantly higher than that of Nanocall as well. Moreover, Nanocall is incapable of calling homopolymer stretches longer than its k-mer length, while current Metrichor basecallers are able to do so. Therefore, as the authors currently state on the Nanocall Github, it should be seen as a platform for testing out novel ideas, rather than a prime choice for basecalling MinION data.\n\nDeepNano Before Metrichor made its own switch from HMM- to RNN-basecalling, the open-source basecaller DeepNano7 already implemented a form of RNN-basecalling, booking a significant improvement in accuracy with respect to the then-current Metrichor version (late 2015). DeepNano was written in Python, using the Theano library38.\n\nRather than LSTM-nodes, as currently used in Metrichor basecallers, DeepNano implements gated recurrent units (GRUs)39. Although the design of GRUs is simpler than that of LSTM units, performance assessments show that GRUs are able to achieve similar or slightly better accuracy in different learning tasks40,41.\n\nDeepNano’s RNN was trained separately for (hairpin-connected) 1D basecalling and true 2D basecalling. As the exact alignment to the reference may be uncertain, the algorithm alternately trains the RNN weights for 100 iterations and then re-aligns the reads to the reference. At the moment of its release in March of 2016, DeepNano was reported by its authors to make more accurate calls than the Metrichor agent for both 1D calling (approximately 70% versus 77%) and 2D reads (approximately 88% versus 87%). However, as the Metrichor agent has since then switched to an RNN-approach as well, these comparisons are outdated.\n\nIn the assembly stage, basecalled sequences are combined to reconstruct continuous parts of the genome as accurately and completely as possible. Assembly of nanopore reads requires a different approach than that of SGS reads; as nanopore reads are longer, finding a correct overlap should be easier, yet they are more error-prone, which increases the uncertainty of overlaps. Because of these differences, a return of interest in overlap layout consensus (OLC) algorithms - which were at the peak of their popularity in the era of Sanger sequencing - is seen. De-Bruijn graph (DBG) assemblers, the more popular choice for SGS reads, were reported to return lower quality assemblies of nanopore reads than OLC-based methods, but proved faster in some cases42. A more detailed comparison between OLC and DBG assemblers, along with implementations of both approaches is discussed in this section. Software using traditional greedy extension algorithms (e.g. SSAKE) was found to perform decidedly less well in a de novo assembly setting, both in terms of assembly quality and required computational resources42, and is therefore omitted here.\n\nPBcR & Canu Originally developed for the first human genome draft, the Celera assembly pipeline43 and its extensions [Koren et al., 201729, Miller et al., 200844, Zimin, 201345, i.a.] have remained a popular choice in a growing landscape of OLC assemblers. Briefly, the Celera assembler uses read overlaps to find contigs of which the structure can unambiguously be derived from overlap information, referred to as unitigs. It then separates repetitive unitigs from unique ones and attempts to orient the unique unitigs with respect to each-other. Where possible, gaps between unique unitigs are filled with repetitive elements. As a high read error rate is detrimental to the quality of the assembly46, two different modifications to the pipeline are available. The PacBio corrected Reads (PBcR) algorithm, originally developed for the correction of PacBio reads suffering from similar error rates, uses accurate short reads mapped with high confidence to the long reads to correct errors. The assembly then proceeds as is usually done by Celera47. PBcR’s successor, Canu29, provides a solution that is more integrated with Celera and does not require short accurate reads. The pipeline includes three stages; correction, trimming and assembly. Overlaps are found using the efficient MHAP28, which hashes k-mers using different hash functions and for each hash function stores the smallest integer to which a k-mer of the sequence is hashed. Comparing the hashed k-mers per read results in initial overlap hits, which are then used to perform error correction by consensus seeking. By selecting overlaps for correction on quality, but limiting the number of overlaps a read can contribute to, Canu attempts to prevent masking of true repeat variants. Shorter reads are used at this stage to improve accuracy of longer reads. In the trimming step, overlaps are recalculated to locate and filter out regions of low coverage and high error. Reads are overlapped two more times to correct specific types of errors (i.e. missed hairpin sections/adapters, chimeric reads) and adjust error rate per overlap, before the actual assembly phase starts. With adjustments to account for erroneous alignments and residual errors, assembly essentially follows the same procedure as CABOG, another Celera-based pipeline44.\n\nDue to its thorough yet relatively efficient correction steps, Canu is significantly more accurate than both its predecessor Celera/PBcR and Minimap/Miniasm25. In a benchmarking effort on Enterobacter kobei reads, it produced an assembly of higher contiguity, with fewer indels and mismatches. This is in line with the accuracy assessment by its authors29.\n\nMinimap & Miniasm In terms of speed and computational efficiency, the OLC-based pipeline consisting of Minimap and Miniasm32 has a definite advantage over other existing tools25. This efficiency was reached through the omission of the consensus step and the use of minimizers. Much like the output of the MinHash algorithm used by Canu29, a minimizer is a memory-efficient hashed representation of a sequence. Minimap computes the set of minimizers of a sequence, the “sketch”, by finding the k-mers represented by the smallest hash value within a certain window size of each position of the sequence (Figure 7). The inverse of each k-mer is also considered. Decreasing the window size will increase the returned number of minimizers and allow for more accurate alignment at the cost of increased computational requirements. Minimap then performs all-versus-all mapping by identifying hits between minimizers of different sequences. The found overlaps are passed on to Miniasm, which constructs an assembly graph. First, artefacts are trimmed from each read by removing all sequences outside the longest stretch with more than 3 times coverage. Then reads contained within other reads are removed and small bubbles, less than 50 kb in length, are popped (i.e. a consensus is taken in cases where paths split and later join up again). Finally, sequences can be extracted from stretches of the graph without multi-edges to form unitigs. The error rate at this point is practically the same as that in the raw reads, emphasizing that correct basecalling is essential for the eventual quality of the assembly. The graphical fragment assembly (GFA) output format of Miniasm conveniently allows both graphing of the uncorrected assembly and addition of consensus error correction tools, such as Nanopolish or Racon, to the pipeline, though the latter increases walltime and computational cost severely25.\n\n(A) To extract one minimizer m, all k-mers and their complements are listed and hashed to a distinct integer using hash function φ. The smallest hash value is then stored with its starting position and a binary value denoting the strand on which it was found (0 for forward and 1 for reverse). If a multiple of the smallest hash value is found within a window, all are stored. (B) The minimizers of all windows in the read are collected and stored as the sketch, M. As M is a set, double minimizers are removed.\n\nIn March of 2016, the authors of Minimap and Miniasm reported assembly of MinION reads of an E. coli genome in a single contig. In May of the same year, Judge et al. assembled an Enterobacter kobei genome in 16 contigs with an N50 of 662 kbase in two minutes, while the next fastest assembler (Canu) took two hours. However their benchmark showed that the omission of an error correction step caused the eventual assembly quality of E. kobei to be too low to properly assess by the QUAST analysis tool25. The inclusion of the Nanopolish consensus correction tool after Miniasm improved the quality of the assembly enough to perform a reliable quality analysis. However, the pipeline was still under-performing compared to other tools.\n\nTULIP As more reads are required to cover larger genomes, and as the time required for all-vs-all overlapping increases quadratically with an increasing number of reads, it follows that the overlap step of OLC assemblers may take unfeasibly long for very large genomes. To tackle this issue, The Uncorrected Long read Integration Process (TULIP) takes a different approach to read overlapping20. Instead of all-vs-all alignment, short seed sequences are selected, which the assembler then attempts to align with long reads. This drastically cuts down the overlapping complexity and makes efficient use of long reads to cover long stretches of the genome between the seed regions. The resulting graph represents seeds as vertices and the connecting reads as edges. In a graph cleaning step, vertices with multiple in- or outgoing edges are revisited. Spurious and superfluous edges are removed aggressively, thus producing a linear graph. Note that, as the name implies, TULIP does not perform basecalling error correction.\n\nThe success of assembly using TULIP highly depends on proper seed selection. To avoid spurious connections between reads, the seeds need to be sufficiently unique in the genome and contain few sequencing errors. If available, SGS reads may be used to construct seeds, although with the increasing accuracy of MinION reads, the ends of long reads may be used as well. Apart from cutting out the need for SGS methods, the latter approach has the added advantage that pairs of seeds are connected by at least one long read. Furthermore, as TULIP is not able to assemble regions in which the gap between seeds is larger than the read length, a proper seed density over the entire genome is required. If a marker map is available for the genome, this information can be used to control the distribution of seeds in the selection process.\n\nAs a first demonstration of TULIP’s efficiency, Jansen et al. assembled the genome of the European eel Anguilla anguilla (approximately 850Mbp) with 18x coverage in three hours (excluding sequence polishing), requiring only 4.4GB of RAM and four threads20. The resulting assembly was more continuous than the SGS-based reference genome. As was the case with Minimap/Miniasm however, the current quality of MinION reads combined with the lack of an error correction step necessitates post-assembly correction. The authors further showed that missed seed alignments were the most commonly encountered issue during graph simplification, followed by tangled alignments due to repetitive seeds and spurious alignments. The seeds, constructed from short SGS reads, only underwent selection by uniqueness, which did not lead to an equal distribution over the entire genome; however, density remained high enough for successful assembly. The authors noted that assembly using the tips of MinION reads as seeds proved successful for Escherichia coli genomes, but this has not been attempted for larger genomes yet (personal communication, May 1, 2017).\n\nHINGE Although long reads provide a definite edge when attempting to resolve repeat regions, issues may still occur if not all individual repeats are spanned by at least one whole read. In such cases, HINGE may provide a solution. Rather than attempting to resolve frayed rope structures in the assembly graph afterwards, HINGE preprocesses the reads to separate repeat regions that are entirely spanned by a read (and are thus more easily resolvable) from those that are not, and collapses the latter beforehand30.\n\nFirst, HINGE attempts to identify reads that wholly or partly overlap a repeat region. It does so by performing all-vs-all alignment and then selecting those reads of which a stretch aligns to a proportionally larger number of other reads than the rest of the read. The intuition behind this is that reads from all copies of a repeat region existing in the genome align to each other, thus causing a characteristic abrupt increase in alignments for reads that overlap these repeat regions. Repeat regions covered entirely by at least one read can be easily resolved and are omitted from the following procedure. Of the reads lining the same repeat region, the reads that extend furthest into the repeat region (regardless of the location of the actual copy), are designated “hinges”. In the subsequent greedy extension of the hinges, the path will split at the hinge regions. Like Miniasm, HINGE outputs its assembly in the form of a graph. As its authors show, this is particularly useful for circular genomes.\n\nHINGE provides an elegant solution to long repeat resolution, by separating resolvable regions from unresolvable ones on forehand. Its authors compared HINGE to Miniasm on PacBio reads of 997 circular bacterial genomes and found that overall, HINGE produced a completed genome in more cases than Miniasm could30. Whether the precaution taken by HINGE is necessary is dependent on the genome under consideration and the used reads; if the genome is known to contain repeats longer than most of the reads, the described approach would be justified.\n\nABruijn While more traditional DBG assemblers performed relatively less well than OLC assemblers on assembling long error-prone reads42, the adapted approach taken by the ABruijn assembler has shown more promise31. To account for the high error rate, ABruijn filters all k-mers occurring in the reads by their frequency; if a k-mer occurs relatively few times, it is assumed that it contains basecalling errors and it is removed. Then, k-mers are fused into so-called “solid strings”, sequences that contain no other occurring sequences as substring. The ABruijn graph is then drawn by representing solid strings as vertices and connecting them where connections exist in the reads. The edges are weighted by the number of positions between the first bases of the connected solid strings. The assembler consults the weights in this graph to quickly identify overlaps between reads, allowing to select on a minimum overlap length and maximum overhang length. The assembly graph is constructed by starting with the graph for an arbitrary read and continuously extending it by overlapping it with other reads. ABruijn also includes an error correction routine, during which a best consensus between reads is found by identifying low-error stretches and, in between those stretches, choosing the consensus sequence that maximizes the likelihood of the read sequences.\n\nUnfortunately, the authors of ABruijn evaluated their assembler only on E. coli MinION reads of an older and more error-prone chemistry (R7.3), and compared their assembly to that of a Nanocorrect and Celera pipeline, which is currently considered obsolete. In this comparison ABruijn performs better, producing an error rate of 1.1% versus 1.5% for Nanocorrect/Celera, with the majority of errors being deletions. How ABruijn stacks up against currently popular assemblers optimized for MinION data on reads produced with up-to-date chemistries is unclear. The more extensive assessment on PacBio reads provided by the authors shows a dramatic decrease in error rate in the ABruijn assembly in comparison to Canu’s, but requiring comparable running time.\n\nA number of tools attempt to improve assemblies by remapping reads to the assembly and adapting the assembly to increase local resemblance to the reads. These tools may be essential to use after assemblers that do not include a consensus step themselves, such as Minimap/Miniasm and SMARTdeNovo, but have also frequently been used to polish assemblies produced by the other assemblers.\n\nNanopolish Nanopolish attempts to find an optimal consensus between an assembly and the raw current signal output by the MinION, by iteratively proposing and evaluating small adaptations to the assembly based on the original reads35. The proposal mechanism for adaptations works in two steps. First, reads are aligned to the assembly and the resulting multiple alignment is divided in 50 bp subsequences of the assembly. For each read aligning partly or fully to a subsequence, sections in which 5-mers perfectly align to the assembly are detected. The consensus sequence between each pair of aligning sections is replaced by the aligned read subsequence, creating an initial set of alternative candidate sequences. In the second step, this set is further extended by proposing every possible one-base deletion, insertion and substitution in the previously generated candidate sequences. Of this set, the sequence maximizing the likelihood of observing the raw signal is picked. This process allows Nanopolish to explore a decent number of likely modifications, while remaining computationally tractable.\n\nPolishing an assembly with Nanopolish was found to improve assembly quality, regardless of the assembly tool used. One study on E. coli sequencing data reported that identity to the reference genome rose from 89% to 99% when Nanopolish was applied after Minimap/Miniasm, while improvement after Canu was more modest (98.2% to 99.6%)48. Although it addresses all types of errors, a large part of the increase in accuracy is reached due to the correction of homopolymeric stretches. Despite its efficient searching heuristic of block replacement and mutation, running Nanopolish remains a time-consuming step; on the error-prone assembly produced by the otherwise quick Minimap/Miniasm pipeline, it may take up to a few extra days of CPU time to finish polishing25,48.\n\nRacon Racon corrects MinION assemblies by finding a consensus sequence between reads and the assembly through the construction of partial order alignment (POA) graphs. After alignment of the reads by a mapper of choice (e.g. Minimap or Graphmap), Racon segments the sequence and finds the best alignment between a POA graph of the reads and the assembly. By default, the alignment is performed using the Needleman-Wunsch algorithm, which can align sequence and POA graph with little adaptation (Figure 8). The alignment process is sped up by parallelization. Racon was reported by its authors to be two orders of magnitude faster than the popular (yet currently deprecated) Nanocorrect after assembly of an E. coli genome by Miniasm, albeit not quite as good at diminishing the error rate (to 1.31% versus 0.62% for Nanocorrect). Compared to consensus steps in Falcon49 and Canu29 on that same assembly, Racon remains an order of magnitude faster while producing similar error rates. A closer look at the error rate reveals that the majority of errors are indels, validating the need for homopolymer and tandem repeat corrections even in a pipeline containing a polishing step with Racon. Finally, the total genome size estimate following application of Racon was closer to the reference genome size than the estimates of Canu, Falcon and Nanocorrect.\n\nIn the latter case, the graph dictates which steps in the matrix are allowed. Taken from Jonathan Dursi’s blog on POA multiple alignment, with permission.\n\n\n3 Discussion\n\nNanopore sequencing is a promising new venue in biology research. Inexpensive, small, capable of producing long reads and freed from the need for nucleotide labeling or amplification, it is conceivable that the MinION will make cost-effective, fast and portable de novo whole genome sequencing of even complex genomes possible in the future. In this review, an attempt was made to give an updated overview of the progress in this field, focusing in particular on de novo whole genome sequencing.\n\nAvailable basecaller tools have been improving rapidly in accuracy. A notable recent improvement was made through the application of RNNs. For the next step in a typical sequencing routine, assembly, OLC-assemblers are currently considered the best option for accurate nanopore-based assembly. The choice of assembler should be adapted to the characteristics of the genome and the priorities of the user. Canu is a complete and accurate solution, while maintaining reasonable CPU times. However, for an assembler without included error correction, Minimap/Miniasm is able to produce a decent assembly and, without any post-assembly correction, is the fastest option available on smaller genomes. For large, complex genomes, TULIP may be the more tractable alternative. Lastly, consensus error correction tools are currently highly useful. Notably, Racon provides a computationally more attractive alternative to the often-used Nanopolish.\n\nCurrently, the most prominent obstacle for de novo sequencing using the MinION is the high error rate of the reads. Improving basecalling accuracy would not only improve assembly quality in a direct manner, but may also allow more computationally efficient assembly.\n\nThe active research community surrounding the MinION has booked great progress in both the development of new applications and improvements on accuracy of existing ones. ONT also continuously works on improvements for both its hardware and software platforms, and regularly updates its users on this.\n\nAlthough these updates often entail welcome new features or some form of accuracy improvement, it should be noted that this policy has also lead to some difficulties. Developers may not be able to keep pace with ONT when evaluating, updating or calibrating their tools, and users may not always know which tool is suited best to their data and needs. As a result, most published studies, including tool benchmarking efforts, were conducted using older or multiple chemistries. Although such growing pains are to be expected for a novel fast-developing field of research, the MinION’s current state of development may allow for some increase in stability, thus giving the user community the time for proper evaluation.\n\n\nNotes\n\niEstimate based on a purchase of 24 flowcells and a 1D/1D2 sequencing kit, 18th of May 2017\n\niiA thorough discussion of RNN architectures and their respective properties is outside the scope of this article. Interested readers are referred to 34 for an introduction to the subject.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nGiovanni Maglia (University of Groningen, Groningen, The Netherlands) provided helpful advice related to the physical basis of nanopore sequencing.\n\n\nReferences\n\nvan Dijk EL, Auger H, Jaszczyszyn Y, et al.: Ten years of next-generation sequencing technology. Trends Genet. 2014; 30(9): 418–426. PubMed Abstract \| Publisher Full Text\n\nDeamer D, Akeson M, Branton D: Three decades of nanopore sequencing. Nat Biotechnol. 2016; 34(5): 518–524. PubMed Abstract \| Publisher Full Text\n\nJain M, Koren S, Quick J, et al.: Nanopore sequencing and assembly of a human genome with ultra-long reads. bioRxiv. 2017. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMyers EW, Sutton GG, Delcher AL, et al.: A whole-genome assembly of drosophila. Science. 2000; 287(5461): 2196–2204. PubMed Abstract \| Publisher Full Text\n\nMiller JR, Delcher AL, Koren S, et al.: Aggressive assembly of pyrosequencing reads with mates. Bioinformatics. 2008; 24(24): 2818–2824. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZimin A: Msr-ca–efficient de novo genome assembler for long and short read data. In Plant and Animal Genome XXI Conference. Plant and Animal Genome. 2013. Reference Source\n\nSalzberg SL, Phillippy AM, Zimin A, et al.: Gage: A critical evaluation of genome assemblies and assembly algorithms. Genome Res. 2012; 22(3): 557–567. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKoren S, Schatz MC, Walenz BP, et al.: Hybrid error correction and de novo assembly of single-molecule sequencing reads. Nat Biotechnol. 2012; 30(7): 693–700. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLu H, Giordano F, Ning Z: Oxford Nanopore MinION Sequencing and Genome Assembly. Genomics Proteomics Bioinformatics. 2016; 14(5): 265–279. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChin CS, Peluso P, Sedlazeck FJ, et al.: Phased diploid genome assembly with single-molecule real-time sequencing. Nat Methods. 2016; 13(12): 1050–1054. PubMed Abstract \| Publisher Full Text" }	[ { "id": "24090", "date": "10 Jul 2017", "name": "David A. Eccles", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nCarlos de Lannoy, Dick de Ridder, and Judith Risse have written a technical review paper of MinION sequencing. This is a challenging task due to the high rate of MinION consumable obsolescence, and the breadth of use cases. Unfortunately I feel that this challenge has not been met in their paper, which appears to be a scattered mixture of discussions of differing recency with no cohesive story threading them together. I think it is especially important that a review paper of MinION technology is as current as possible, because it has the potential to be used extensively by others as an indication of the quality of sequencing at a particular point in time.\nI should perhaps point out that the main thing I found the most frustrating to read was a reference to R9 as current technology, and my other comments are likely coloured by me spotting this in my first scan through the article.\nHere are some additional comments about the content of the paper. This is not a comprehensive review, and should only be used as a guide to the areas of concern I have with the content:\nTitle: \"Coming of age\" -- overlaps with Sara Goodwin's Nature Reviews Genetics paper [doi:10.1038/nrg.2016.49], which is also a review paper on sequencing technology (including nanopore). See, for example, this image:\n[https://www.nature.com/nrg/journal/v17/n6/fig_tab/nrg.2016.49_F5.html]\nA search for \"coming of age\" and nanopore returns another paper:\n[http://pubs.rsc.org/en/content/articlelanding/2007/mb/b702845h]\n\nThe paper talks about \"current\" nanopore technology as R9, which was discontinued last year in preference to R9.4. Shortly before this, the paper discusses solid state nanopores for a review paper, when there is no commercially-available product. This is despite mentioning the introduction of 1D² technology in May 2017, which was introduced after R9.4 pores.\n\nThe paper references internal nanopore community technical documentation, which is unlikely to be available to general readers of the article (and particularly not those who are considering its use).\n\n\"The exact start and end of the hairpin is not always clear from the signal\" -- I disagree with this. While current software may have difficulty interpreting the start/end, the location is obvious when looking at the raw signal. It is surprising to see this shortly before referencing our chimeric reads paper, where we give visual examples of the raw signal where the characteristic harpins are annotated.\n\nFigure 5: I like the idea of this figure. It's uncommon for people to give a visual indication of multiple pores and the MUX switching. However, some changes (and/or different annotation) are warranted. Using colours for both the mux type and the QC error is distracting; I would recommend using a hatch pattern to indicate failed pores. The pores are much larger than their actual size relative to the well size; this should be stated in the legend. Pores can also fail QC due to the membrane breaking, due to blocked pores, and presumably other reasons; the QC check is not only counting pores.\n\nFigure 6: There is enough event-annotated nanopore data available that mock data should not be necessary. Spikes and within-event signal variation that I am used to seeing in raw signal data are not present in this figure.\n\n\"Any comparison in this chapter\" -- might want to change that 'chapter' to a 'paper'.\n\nCanu is now published in Genome Research:\n[dx.doi.org/10.1101/gr.215087.116] The nanopolish algorithm has now been improved such that it takes substantially less time for v0.7:\n[http://simpsonlab.github.io/2017/06/30/nanopolish-v0.7.0/] The paper makes many statements around the accuracy of base-calling and assembly solutions which [mostly] ignores post-transducer base-calling. I would like to see a review paper that (if possible) concentrates on post-transducer software, which would probably mean discarding discussion on HMM-based callers.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? No\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Partly", "responses": [ { "c_id": "2871", "date": "12 Jul 2017", "name": "Carlos de Lannoy", "role": "Author Response", "response": "Thank you for this thorough review. It clearly raises a number of valid points, which we will address in a revised version after other reviews have become available. We would like to point out at this time that we did intend our review to cover the most recent work, and that by “R9” we actually meant “R9.x” (R9.0-R9.5), as opposed to R7. This also explains why indeed we discuss more recent developments than R9.0, such as ID^2 sequencing. We hope this takes away some of the issues." }, { "c_id": "2873", "date": "13 Jul 2017", "name": "David Eccles", "role": "Reviewer Response", "response": "\"we did intend our review to cover the most recent work\"Note that the author guidelines for Review articles state that \"Reviews should provide a balanced and comprehensive overview of the latest discoveries in a particular field.\" See here. This is what I had in my mind in my decision for Approve/Reject.It is particularly important for nanopore reviews that this is the case because ONT advances their technology and software at a very rapid rate. Academics who decide on funding for research frequently latch onto the latest review articles, as they expect those articles to be a comprehensive representation of the current state of the technology." } ] }, { "id": "24095", "date": "25 Jul 2017", "name": "Christiaan V. Henkel", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper by Carlos de Lannoy et al. is a timely review of the choices available for genome sequencing based on Oxford Nanopore technology. This is a fast-moving field, and potential entrants will be well served by this exposition of the many options available. However, precisely because it is a fast-moving field, several of these options are already obsolete (yet relevant in providing context for recent developments). In addition, the many options in bioinformatics are confounded with a multiplicity of genome types (small prokaryotic to large repetitive) and nanopore chemistries.\nMany readers would benefit from additional support in navigating these multiple menageries. For this reason, we would like to ask the authors to address the following issues in more detail:\nAll methods discussed have been tested using nanopore data, but never using comparable datasets. Therefore, evaluating for example the percentages of sequence correctness is not straightforward. A table simply listing the methods with some of their key characteristics/assembly statistics/nanopore chemistries/genomes assembled would be helpful.\nSimilar confusion is always looming because of the many nanopore chemistries that have been available. For example, on page 7, on the improvement of the Metrichor basecallers: the improvement here, from 68% to 95%, also coincides with the shift from R7.3 to R9. While this is mentioned as ‘numerous improvements in chemistry and hardware’, it would be clearer if the nanopore chemistry generation is explicitly mentioned whenever such percentages are listed.\n\nThe in-depth discussion of assembly methodology is clearly delimited to methods focusing on nanopore data only. But is this actually already a feasible option, and if not, what is needed to make this happen? All of the assembly pipelines discussed still fall short of reference genome quality. At the moment, sequence correction using short-read data (Pilon) remains necessary. Why/when should one choose for nanopore-only assembly, as opposed to hybrid?\n\nBoth basecalling and polishing are nanopore-specific bioinformatics tasks, however the de novo assemblers are in principle (and often in practice) suitable for any long-read technology. Therefore, a brief discussion comparing PacBio and nanopore for genome assembly is appropriate. PacBio-specific assemblers are briefly mentioned (SMARTdenovo, Falcon), but how do they relate to e.g. Canu?\n\nA few minor issues:\np2, p6: please change ‘chapter’ to ‘paper’.\n\np3, second column, modifications of the R9/CsgG pore: this is a good location to briefly introduce R9.4/R9.5\n\np5: ‘multiple reads may be linked together by hairpins into chimeric reads’ -> multiple molecules\n\nFigure 5: explain that the white wells are ignored.\n\np8, Canu: ‘Where possible, gaps between unique unitigs are filled with repetitive elements.’ This may need some rephrasing, ‘repetitive element’ is a specific term – not necessarily identical with ‘non-unique genome content’.\n\np8: percentages for DeepNano: ‘more accurate … (approximately 70% versus 77%) and … (approximately 88% versus 87%)’. Which is which?\n\nFigure 7, 3rd kmer: the reverse complement of CTC isn’t TCT.\n\np11: a few extra days of CPU time spent on polishing is acceptable for large genomes, but a major bottleneck for bacteria – polishing then takes longer than sequencing. Do these timings refer to prokaryotic genomes?\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-1083
https://f1000research.com/articles/6-1977/v1	08 Nov 17	{ "type": "Research Article", "title": "The decision to delivery interval in emergency caesarean sections: Impact of anaesthetic technique and work shift", "authors": [ "Anette Hein", "David Thalen", "Ylva Eriksson", "Jan G. Jakobsson", "Anette Hein", "David Thalen", "Ylva Eriksson" ], "abstract": "Background: One important task of the emergency anaesthesia service is to provide rapid, safe and effective anaesthesia for emergency caesarean sections (ECS). A Decision to Delivery Interval (DDI) <30 minutes for ECS is a quality indicator for this service. The aim of this study was to assess the DDI and the impact of chosen anaesthetic technique (general anaesthesia (GA), spinal anaesthesia (SPA) with opioid supplementation, or “top-up” of labour epidural analgesia (tEDA) with local anaesthesia and fentanyl mixture) and work shift for ECS at Danderyds Hospital, Sweden. Methods: A retrospective chart review of ECS at Danderyds Hospital was performed between January and October 2016. Time between decision for CS, start of anaesthesia, time for incision and delivery, type of anaesthetic technique, and time of day, working hours or on call and day of week, Monday – Friday, and weekend was compiled and analysed. Time events are presented as mean ± standard deviation. Non-parametric tests were used. Results: In total, 135 ECS were analysed: 92% of the cases were delivered within 30 minutes and mean DDI for all cases was 17.3±8.1 minutes. GA shortened the DDI by 10 and 13 minutes compared to SPA and tEDA (p<0.0005). DDI for SPA and tEDA did not differ. There was no difference in DDI regarding time of day or weekday. Apgar <7 at 5’ was more commonly seen in ECS having GA (11 out of 64) compared to SPA (2/30) and tEDA (1/41) (p<0.05). Conclusion: GA shortens the DDI for ECS, but the use of SPA as well as tEDA with opioid supplementation maintains a short DDI and should be considered when time allows. Top-up epidural did not prolong the DDI compared to SPA. The day of week or time of ECS had no influence on the anaesthesia service as measured by the DDI.", "keywords": [ "Caesarean section", "anaesthesia", "time factors" ], "content": "Introduction\n\nThere are around 110,000 births annually in Sweden, and the national statistics shows a trend for an increasing number of caesarean sections (CS). In 2014, 17% of all births in Sweden were CSi. CS may be divided into emergency and elective procedures. Emergency CS (ECS) are commonly defined as follows: to be performed within an adequate time frame to avoid negative effects on neonate and/or mother, while elective are performed where there is no time constrain. Lucas four graded scale categorize CS by degree of urgency, as follows: 1) immediate threat to life of woman or foetus; 2) maternal or foetal compromise that is not immediately life-threatening; 3) needing early delivery but no maternal or foetal compromise; and 4) at a time to suite patient and maternity team1. Dupuis suggested a coloured system to distinguish grade of emergency, to facilitate the communication, thus facilitating the process and shorten the DDI2.\n\nNeed for an urgent CS is among the most dramatic anaesthetic events, requiring effective and vigilant services. It has been suggested that neonates should be delivered within 20 to 30 minutes after the decision of an urgent CS has been made3. The time interval is, however, an extrapolation around time for the development of serious, life threatening acid base compromise4. Various logistical programs aiming at improving the service have shown that time between decision and delivery can be reduced5. The Swedish Society for Anaesthesia & Intensive Care has set recommendations that anaesthetic services should include an anaesthesiologist available within 5 minutes from the decision of an obstetric emergency and that an emergency CS incision should be possible to start within 15 minutes from the decisionii. The explicit evidence to support a clear medical benefit of the 30 minute decision to delivery interval (DDI) limit is sparse and this may be more of a tool to use for auditing of anaesthesia services6. The NICE guidelines extend category 2: Perform category 2 CS in most situations within 75 minutes of making the decisioniii. It should also be acknowledged that the DDI is a composite end-point, including delay between obstetric decision, press the alarm button, time until start of anaesthesia, anaesthesia ready for surgery, and surgery time.\n\nThe primary aim of the present study was to assess the impact of anaesthetic technique and work shift on the DDI in emergency CS, with a decided DDI <30 minutes at our hospital, Danderyds Hospital (category 1 and emergency category 2 CS).\n\n\nMethods\n\nThe study was reviewed and approved by the Stockholm Ethical Review Board (reference number: 2016/825-31).\n\nThis is a retrospective chart review study using a proforma protocol; alarm logs and patient records for ECS at Danderyds Hospital from 1st of January to October 31st 2016 were collected and analysed. From the alarm logs, we assessed the time of the event and we then matched this information with the performed CS in our electronic surgical registration system (Orbit 5.7), from where we retrieved start time of anaesthesia, start and end of operation and type of anaesthesia performed. The follow up regarding foetal status and need of treatment was collected from the patient journal.\n\nDanderyds Hospital is an emergency hospital with about 530 beds for general and gynaecological surgery, medicine and cardiac clinic, and includes two delivery departments with a total of 10800 deliveries/year. Two anaesthesia specialists and one anaesthesia registrar are in house on call for all anaesthesia services, including intensive care. There are at minimum three surgical teams, each including one anaesthesia nurse, one surgical nurse and one or two nursing assistants. One surgical team is located in and reserved for the women’s surgical department. In case of obstetric emergency collisions, a team from the general surgical department (located in the same building) reach the women’s department in 1–2 minutes to assist.\n\nWhen the attending obstetrician decides on ECS in the most severe cases, needing immediate delivery, the alarm is pressed gathering the surgical team together with anaesthesia specialist, anaesthesia registrar and neonatologist. When the obstetrician estimates the ECS need a DDI within 30 minutes, the obstetrician first calls the anaesthesia specialist by phone to give a short report, including whether there is a well working epidural to top-up and then presses the alarm to gather the team. The obstetrician follows the patient to facilitate the process.\n\nThe OR is located central of the largest delivery department on the same floor and close to the women’s surgical department – within reach in 30–60 seconds. When occupied, an OR in the women’s surgical department is used.\n\nWhen an ECS with an estimated DDI time >30 minutes is decided, the surgical team is gathered by phone and no alarm is used.\n\nThe anaesthesiologist specialist on call, responsible for the obstetric anaesthesia services decides on anaesthetic technique per set routines at the department. When the obstetrician urges for an immediate delivery general anaesthesia (GA) is recommended in most cases by our routines and with an explicit need for delivery within 30 minutes’ regional anaesthesia (RA) is recommended.\n\nIn the present study, only ECS needing the alarm, delivery immediately and up to 30 minutes, were studied, with the most common cause being sign of foetal distress.\n\nNo intervention or change of routine was initiated by or associated with the present study. GA was based on a rapid sequence induction with propofol and suxamethonium following pre-oxygenation and sevoflurane until umbilical cord is divided. Neuroaxial anaesthesia, spinal and top-up epidural followed standard routines. Spinal anaesthesia (SPA) with bupivacaine (approximately 2.4 ml 5 mg/ml), morphine (100 µg) and fentanyl (10 µg); top-up epidural (tEDA) with ropivacaine (7.5 mg/ml) and fentanyl (100 µg/20 ml ropivacaine) 15 – 20 ml.\n\nDescriptive statistics regarding the ECS and the variables in this study was made using mean, standard deviation (SD) and range, as well as median and interquartile range (IQR), as appropriate. Mann-Whitney U test was used for comparing means between two variables and Kruskal-Wallis H test was used when comparing two or more groups. Chi-square-test was used for test of differences between category data. A p-value <0.05 was considered significant and all data were analysed in IBM SPSS Statistics 23.\n\n\nResults\n\nDuring the study period, 150 ECS were identified from the record systems. Data for analysis was not retrieved in 15 cases, thus 135 ECS were included in the present analysis (Figure 1).\n\nThe median DDI for the 135 ECS studied was 17 minutes (range, 5–41). The median DDI was significantly shorter for GA (10 minutes) compared to RA (SPA 20 minutes, tEDA 23 minutes) (p<0.0005; Table 1). The major time difference was in the time to establish adequate anaesthesia; GA shortened anaesthesia start to ready for surgery by 7 and 8 minutes as compared to SPA and tEDA, respectively. The time difference between SPA and tEDA was 1 minute: total 9 minutes (range, 1–16) and 10 minutes (range, 1–23), respectively (Table 1).\n\nData are presented in minutes as median (range).\n\n*** P < 0.0005 compared to reginal anaesthesia, ns. No significant difference between SPA and tEDA. GA, general anaesthesia; SPA, spinal anaesthesia; tEDA, top-up epidural anaesthesia; DDI, decision to delivery interval.\n\nThere was no significant difference in DDI between different working shifts: daytime, on call during the week and weekends (Table 2, Figure 2). Also, when divided into different time events, call to start of anaesthesia, start of anaesthesia to surgery, and surgery to delivery, no statistical difference between the different working shifts was found.\n\nData are presented in minutes as median (range).\n\nDDI, decision to delivery interval.\n\nGA, general anaesthesia; SPA, spinal anaesthesia; tEDA, top-up epidural anaesthesia; DDI, decision to delivery interval.\n\nFourteen neonates had an Apgar score of <7 at 5 minutes: 11 out of the 64 mothers that received GA, 2 out of the 30 that received SA and 1 out of the 42 that received tEDA (p < 0.05). In all, 39 neonates were transferred to the neonatal intensive care for further observation and treatment: 22, 10 and 7 had GA, SPA and tEDA, respectively (ns.; Table 3).\n\n* p<0.05 Chi-square test, ns none significant between anaesthetic techniques\n\nGA, general anaesthesia; SPA, spinal anaesthesia; tEDA, top-up epidural anaesthesia; Apgar 5’, Apgar score at five minutes; CPAP, continuous positive airway pressure\n\n\nDiscussion\n\nOur study was designed as a quality audit of an important part of our anaesthesia service, providing effective anaesthesia for ECS. Our anaesthesia service was seemingly effective: work shift and day of the week did not impact the DDI. General anaesthesia was expectedly associated with the shortest time for anaesthesia, as well as the lowest DDI; however the DDI was kept within 30 minutes in a clear majority of cases also when spinal anaesthesia and top-up epidural anaesthesia were chosen. The conversion of an established labour epidural, increased time for anaesthesia and DDI, but only marginally. We did not find that the use of spinal anaesthesia or top-up epidural worsened neonate outcome. Thus, we do consider that our anaesthetic service is in line with national and local guidelines, since time to establish surgical anaesthesia was achieved in a timely fashion 24/7.\n\nTime recommendations, such as a 30 minute DDI for ECS is more of a general recommendation than based on firm evidence. Anaesthesia for CS should always be managed on a benefit vs. risk basis. The degree of foetal and or maternal distress should form the basis for management and haste of delivery. One of our primary aims was to assess how our emergency obstetric anaesthesia service performed and thus an analysis of time lines was found to be a reasonable indicator. In 2006, Blom et al. published the results from a study assessing DDI in the US. Of the included 11,481 CS, 2,808 were performed for an emergency indication7. Of these, 1,814 (65%) began within 30 minutes of the decision to operate, thus a lower figure than ours. Likewise, in a more recent meta-analyses, Tolcher et al. found that 79% of category 1 deliveries and 36% of category 2 deliveries were achieved within 30 minutes, with significantly shorter time in category 1 compared to category 2 deliveries8. Thus, our service was found effective and “superior” to the results found in that study.\n\nTime is of course not the key important variable; neonatal and parental outcome is without doubt the most important outcome. However, the aim of the present study was to assess logistics and the quality of the anaesthesia services. The proportion of neonates with a low 5 minute Apgar score was higher among the mothers that received GA. We interpret this finding as the result of the intra-uterine distress and not to the anaesthetic choice per se. We cannot, unfortunately, explicitly describe the degree of foetal distress, nor any further information around obstetric factors, placental ablatio, vaginal bleed, foetal Ph, etc. Blom et al. found that new-borns showing compromise, such as umbilical artery pH less than 7 and intubation in the delivery room, were significantly greater when the CS was commenced within 30 minutes, likely attesting to the need for emergent delivery. This is in line with our observations. Studies investigating neonatal outcomes correlated to DDI showed that there was a higher risk of overall 5-minute Apgar score < 7 and umbilical artery pH level <7.10 in cases involving shorter DDI. A study from Singapore published in 2016 showed results similar to ours; general anaesthesia was associated with a shorter DDI, but worse perinatal outcomes than regional anaesthesia9. It must be acknowledged that this is a retrospective observational study. We did by no means intervene with what technique should be used. Anaesthesia was solely chosen by the anaesthetist on basis on the urgency for delivery.\n\nWe did not find any major difference in time delay between spinal anaesthesia, combining bupivacaine, fentanyl and morphine, and top-up epidural combining ropivacaine and fentanyl regarding time or neonatal outcome. Strouch et al. studied neonatal acid-base status and did not find any further acidosis associated to conversion epidural compared to spinal anaesthesia10. We used a bupivacaine, morphine and fentanyl combination for the spinal anaesthesia, and ropivacaine and fentanyl for the epidural top-up. We used the 100-µg intrathecal dose morphine since it has been suggested to be an adequate balance between its benefits and side effects, pruritus and nausea/vomiting11. Fentanyl facilitates onset12–14 and improves intraoperative analgesia. The addition of fentanyl for epidural anaesthesia has also been shown to improve quality of anaesthesia15,16. However, the intraoperative effect has been discussed for elective CS17.\n\nThe conversion of a labour epidural to regional anaesthesia suitable for CS has been debated, but is today seemingly well-accepted practice18,19. The success rate for conversion is high; however prolonged duration of labour analgesia, repeated need of clinician administered bolus doses and obesity are factors suggested to increase the risk of failure20,21. Lidocaine with adrenalin with fentanyl supplementation is commonly used for conversion18. Allam et al. showed carbonated lidocaine with adrenaline to be twice as fast as sole levo-bupivacaine to achieve a T5 touch/T4 cold block, when used for conversion22. Carbonated local anaesthetics are not available in Sweden. A previous study comparing lidocaine/adrenaline/fentanyl to plain bupivacaine did not show significant difference in time to be ready for surgery23. Sng et al. compared 2% lignocaine with adrenaline and fentanyl, 0.75% ropivacaine and 0.5% levo-bupivacaine for extension of low dose epidural analgesia for urgent CS and did not find any significant difference in time to reach surgical anaesthesia24. We used ropivacaine and fentanyl mixture and achieved a rapid conversion.\n\nThe mother is exposed to increased anaesthetic risk when ECS is performed under general anaesthesia25. Endler et al. suggested, following their review of maternal mortality in 1988, regional anaesthesia to be used when possible, avoiding the risk for serious airway complications26. However, regional anaesthesia is not without risk27,28 and the Cochrane meta-analysis published in 2012 could not show any significant difference between general and regional anaesthesia in terms of risks29.\n\nThe present results must be put into the perspective of the routine at our institution. When a push-button call is made by the obstetrician for a category 1 CS, we have always at least one experienced anaesthesiologist who is called together with anaesthesia and scrub nurses and a neonatologist. We have been working with the communication and process to facilitate regional anaesthesia when that is an option. The obstetrician informs the anaesthesiologist immediately if there is a labour epidural to top-up. The routine is to start the surgery activating dose outside, but close to, the operation theatre when it seems appropriate, with continued supervision by the anaesthesiologist, when a well working labour epidural is in place and mother and child’s status allow. If a well working labour epidural is not in place a rapid spinal is chosen, if there is time. Warm Ringers lactate (1000 ml) is used as co-load and phenylephrine as first line to stabilize blood pressure to be combined with ephedrine where pulse is < 75 min-1. During day time when the regular operation program is on-going there is always one room spared for emergent CS.\n\nThere are several limitations with our study. We have unfortunately not been able to discriminate absolute grade of emergency apart from the attending obstetricians’ decision of a DDI less than 30 minutes in the performed CS, due to our record keeping. The decision for anaesthetic technique may of course have been influenced by the degree of foetal and/or mother compromise. It is common practice to choose GA for the most urgent category 1 ECS, and to reserve spinal and epidural top-up for cases with less maternal or foetal compromise. We found a tendency of less pCO2, lower need of CPAP, ventilation and admission to neonatal unit in favour for EDA vs. GA and even vs. SA. Indeed, epidural top-up might have been chosen for the healthiest foetus. We did furthermore not explicitly study maternal effects, e.g. need for supplementation analgesia during regional anaesthesia. Consequently, further studies are warranted.\n\nTerbutaline administration i.v. is common practice in our intrapartum intra-uterine resuscitation routine in case of asphyxia ECS, but we did not analyse the number of patients receiving tocolytics. Other possible factors are the occurrence of maternal hypotension events and amount consumed vasopressor, ephedrine and phenylephrine, which was not analysed in our study. Foetal heart rate monitoring is continued during the top-up epidural procedure and as foetal heart rate pattern is improved the need of urgency decreases and this might influence the DDI time.\n\nIn conclusion, we found our emergency obstetric anaesthesia service effective and adherent to guidelines for DDI. Anaesthesia for ECS must, however, always be based on an individual assessment, benefit vs. risk for mother and child. General anaesthesia was as expected associated with a more rapid DDI, but spinal anaesthesia with bupivacaine, morphine and fentanyl mixture, as well as top-up labour epidural provided similar rapid time to delivery and seems a reasonable benefit vs. risk option for category 1 ECS with acceptable DDI within 20–30 minutes. Further studies assessing effect on neonatal outcome associated with the choice of anaesthetic technique are warranted.\n\n\nData availability\n\nDataset 1: Raw data for the present study. doi, 10.5256/f1000research.13058.d18353330\n\n\nFoot notes\n\nihttp://www.socialstyrelsen.se/nyheter/2014december/andelenkejsarsnittvarierarkraftigtilandet\n\niihttps://sfai.se/wp-content/uploads/files/11-4%20Obstetrisk_anestesi-och%20intensivv%C3%A5rd_organsation%20.pdf\n\niiihttps://www.nice.org.uk/guidance/cg132/chapter/1-Guidance", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe study has been supported by the department of Anaesthesia, with no external funding.\n\n\nReferences\n\nLucas DN, Yentis SM, Kinsella SM, et al.: Urgency of caesarean section: a new classification. J R Soc Med. 2000; 93(7): 346–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDupuis O, Sayegh I, Decullier E, et al.: Red, orange and green Caesarean sections: a new communication tool for on-call obstetricians. Eur J Obstet Gynecol Reprod Biol. 2008; 140(2): 206–11. PubMed Abstract \| Publisher Full Text\n\nHillemanns P, Strauss A, Hasbargen U, et al.: Crash emergency cesarean section: decision-to-delivery interval under 30 min and its effect on Apgar and umbilical artery pH. Arch Gynecol Obstet. 2005; 273(3): 161–5. PubMed Abstract \| Publisher Full Text\n\nHolcroft CJ, Graham EM, Aina-Mumuney A, et al.: Cord gas analysis, decision-to-delivery interval, and the 30-minute rule for emergency cesareans. J Perinatol. 2005; 25(4): 229–35. PubMed Abstract \| Publisher Full Text\n\nWeiner E, Bar J, Fainstein N, et al.: The effect of a program to shorten the decision-to-delivery interval for emergent cesarean section on maternal and neonatal outcome. Am J Obstet Gynecol. 2014; 210(3): 224.e1–6. PubMed Abstract \| Publisher Full Text\n\nNational Institute for Health and Care Excellence: Caesarean section guideline. London (UK): National Institute for Health and Care Excellence, 2011. Reference Source\n\nBloom SL, Leveno KJ, Spong CY, et al.: Decision-to-incision times and maternal and infant outcomes. Obstet Gynecol. 2006; 108(1): 6–11. PubMed Abstract \| Publisher Full Text\n\nTolcher MC, Johnson RL, El-Nashar SA, et al.: Decision-to-incision time and neonatal outcomes: a systematic review and meta-analysis. Obstet Gynecol. 2014; 123(3): 536–48. PubMed Abstract \| Publisher Full Text\n\nDunn CN, Zhang Q, Sia JT, et al.: Evaluation of timings and outcomes in category-one caesarean sections: A retrospective cohort study. Indian J Anaesth. 2016; 60(8): 546–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStrouch ZY, Dakik CG, White WD, et al.: Anesthetic technique for cesarean delivery and neonatal acid-base status: a retrospective database analysis. Int J Obstet Anesth. 2015; 24(1): 22–9. PubMed Abstract \| Publisher Full Text\n\nSultan P, Halpern SH, Pushpanathan E, et al.: The Effect of Intrathecal Morphine Dose on Outcomes After Elective Cesarean Delivery: A Meta-Analysis. Anesth Analg. 2016; 123(1): 154–64. PubMed Abstract \| Publisher Full Text\n\nDahlgren G, Hultstrand C, Jakobsson J, et al.: Intrathecal sufentanil, fentanyl, or placebo added to bupivacaine for cesarean section. Anesth Analg. 1997; 85(6): 1288–93. PubMed Abstract \| Publisher Full Text\n\nAgrawal A, Asthana V, Sharma JP, et al.: Efficacy of lipophilic vs lipophobic opioids in addition to hyperbaric bupivacaine for patients undergoing lower segment caeserean section. Anesth Essays Res. 2016; 10(3): 420–424. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBozdogan Ozyilkan N, Kocum A, Sener M, et al.: Comparison of intrathecal levobupivacaine combined with sufentanil, fentanyl, or placebo for elective caesarean section: a prospective, randomized, double-blind, controlled study. Curr Ther Res Clin Exp. 2013; 75: 64–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGogarten W: Spinal anaesthesia for obstetrics. Best Pract Res Clin Anaesthesiol. 2003; 17(3): 377–92. PubMed Abstract \| Publisher Full Text\n\nParate LH, Manjrekar SP, Anandaswamy TC, et al.: The effect of addition of low dose fentanyl to epidural bupivacaine (0.5%) in patients undergoing elective caesarean section: A randomized, parallel group, double blind, placebo controlled study. J Postgrad Med. 2015; 61(1): 27–31. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOlofsson C, Ekblom A, Sköldefors E, et al.: Anesthetic quality during cesarean section following subarachnoid or epidural administration of bupivacaine with or without fentanyl. Acta Anaesthesiol Scand. 1997; 41(3): 332–8. PubMed Abstract \| Publisher Full Text\n\nBauer ME, Kountanis JA, Tsen LC, et al.: Risk factors for failed conversion of labor epidural analgesia to cesarean delivery anesthesia: a systematic review and meta-analysis of observational trials. Int J Obstet Anesth. 2012; 21(4): 294–309. PubMed Abstract \| Publisher Full Text\n\nDepuydt E, Van de Velde M: Unplanned cesarean section in parturients with an epidural catheter in-situ: how to obtain surgical anesthesia? Acta Anaesthesiol Belg. 2013; 64(2): 61–74. PubMed Abstract\n\nOrbach-Zinger S, Friedman L, Avramovich A, et al.: Risk factors for failure to extend labor epidural analgesia to epidural anesthesia for Cesarean section. Acta Anaesthesiol Scand. 2006; 50(7): 793–7. PubMed Abstract \| Publisher Full Text\n\nLee S, Lew E, Lim Y, et al.: Failure of augmentation of labor epidural analgesia for intrapartum cesarean delivery: a retrospective review. Anesth Analg. 2009; 108(1): 252–4. PubMed Abstract \| Publisher Full Text\n\nAllam J, Malhotra S, Hemingway C, et al.: Epidural lidocaine-bicarbonate-adrenaline vs levobupivacaine for emergency Caesarean section: a randomised controlled trial. Anaesthesia. 2008; 63(3): 243–9. PubMed Abstract \| Publisher Full Text\n\nGoring-Morris J, Russell IF: A randomised comparison of 0.5% bupivacaine with a lidocaine/epinephrine/fentanyl mixture for epidural top-up for emergency caesarean section after \"low dose\" epidural for labour. Int J Obstet Anesth. 2006; 15(2): 109–14. PubMed Abstract \| Publisher Full Text\n\nSng BL, Pay LL, Sia AT: Comparison of 2% lignocaine with adrenaline and fentanyl, 0.75% ropivacaine and 0.5% levobupivacaine for extension of epidural analgesia for urgent caesarean section after low dose epidural infusion during labour. Anaesth Intensive Care. 2008; 36(5): 659–64. PubMed Abstract\n\nKrom AJ, Cohen Y, Miller JP, et al.: Choice of anaesthesia for category-1 caesarean section in women with anticipated difficult tracheal intubation: the use of decision analysis. Anaesthesia. 2017; 72(2): 156–171. PubMed Abstract \| Publisher Full Text\n\nEndler GC, Mariona FG, Sokol RJ, et al.: Anesthesia-related maternal mortality in Michigan, 1972 to 1984. Am J Obstet Gynecol. 1988; 159(1): 187–93. PubMed Abstract \| Publisher Full Text\n\nHawkins JL, Koonin LM, Palmer SK, et al.: Anesthesia-related deaths during obstetric delivery in the United States, 1979–1990. Anesthesiology. 1997; 86(2): 277–84. PubMed Abstract \| Publisher Full Text\n\nHawkins JL, Chang J, Palmer SK, et al.: Anesthesia-related maternal mortality in the United States: 1979–2002. Obstet Gynecol. 2011; 117(1): 69–74. PubMed Abstract \| Publisher Full Text\n\nAfolabi BB, Lesi FE: Regional versus general anaesthesia for caesarean section. Cochrane Database Syst Rev. 2012; 10: CD004350. PubMed Abstract \| Publisher Full Text\n\nHein A, Thalen D, Eriksson Y, et al.: Dataset 1 in: The decision to delivery interval in emergency caesarean sections: Impact of anaesthetic technique and work shift. F1000Research. 2017. Data Source" }	[ { "id": "27757", "date": "16 Nov 2017", "name": "Antti J. Vaananen", "expertise": [ "Reviewer Expertise Anesthesia for cesarean delivery" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a sound piece of work. Some points to consider regarding the article:\nMethods: The detailed description of the institutional procedures is a merit to this paper as it facilitates comparison of the results to other institutions. Were the parturients allocated into the different anesthesia groups according to the intended anesthesia type or final anesthesia type (is failed epidural top-up resulting in GA analyzed as epidural top-up or GA)? Was the rate of regional anesthesia failures addressed?\n\nRegarding epidural top-ups: The \"start anesthesia to ready for surgery\" is surprisingly fast for the epidural group given that ropivacaine (7.5 mg/ml) is used as the epidural anesthetic. It is stated in the paper that the top-up was initiated at the labour ward before transfer to the operating room which can be considered safe even in the absence of haemodynamic monitoring during transfer IF: a) the attending anesthesiologist is following the parturient to the operating room in these cases and b) the distance (=transfer time) from the labour room to the operating room is not long. It would be helpful for the reader to see a brief description about the underlying labour analgesia system used (continuous infusion vs boluses on demand with or without background infusion) for the parturients with epidural catheters as this may have a major effect on the onset time of epidural top-up.\n\nGeneral comments: As the authors note in the discussion, it is evident that GA is more likely to be chosen in the more urgent cases (evident also as a 2 minutes lower median \"call to start of anesthesia -time\" and worse infant outcome parameters). This underlying difference in the obstetric urgency affects direct comparison of the anesthesia modes on outcome DDI times and could warrant presentation of the data in Table 2 separately for the GA cases as well as for the regional cases (even separately for all three anesthesia subgroups). Ultimately this study provides important aspects in relation to the very similar overall DDI time when either spinal anesthesia or epidural anesthesia is employed (Table 1 and Figure 2) while showing that GA results in superior outcome DDI times - at least when obstetric emergency requires.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3245", "date": "11 Dec 2017", "name": "Jan Jakobsson", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Referees. Thank you for effective read and constructive and important comments. We have tried to amend the paper accordingly below are explicit responses to your comments. Responses in ItalicsMethods:The detailed description of the institutional procedures is a merit to this paper as it facilitates comparison of the results to other institutions.Were the parturients allocated into the different anesthesia groups according to the intended anesthesia type or final anesthesia type (is failed epidural top-up resulting in GA analyzed as epidural top-up or GA)? Was the rate of regional anesthesia failures addressed?We did not have failure of blocks explicitly registered, thus we can’t give any exact data/details.Regarding epidural top-ups:The \"start anesthesia to ready for surgery\" is surprisingly fast for the epidural group given that ropivacaine (7.5 mg/ml) is used as the epidural anesthetic. It is stated in the paper that the top-up was initiated at the labour ward before transfer to the operating room which can be considered safe even in the absence of haemodynamic monitoring during transfer IF: a) the attending anesthesiologist is following the parturient to the operating room in these casesAdded to discussion, and b) the distance (=transfer time) from the labour room to the operating room is not long. Added in discussion,It would be helpful for the reader to see a brief description about the underlying labour analgesia system usedAdded in the discussion (continuous infusion vs boluses on demand with or without background infusion) for the parturients with epidural catheters as this may have a major effect on the onset time of epidural top-up. General comments:As the authors note in the discussion, it is evident that GA is more likely to be chosen in the more urgent cases (evident also as a 2 minutes lower median \"call to start of anesthesia -time\" and worse infant outcome parameters). This underlying difference in the obstetric urgency affects direct comparison of the anesthesia modes on outcome DDI times and could warrant presentation of the data in Table 2 separately for the GA cases as well as for the regional cases (even separately for all three anesthesia subgroups). Ultimately this study provides important aspects in relation to the very similar overall DDI time when either spinal anesthesia or epidural anesthesia is employed (Table 1 and Figure 2) while showing that GA results in superior outcome DDI times - at least when obstetric emergency requires.Thank you for the adequate comment, the outcome is in our eyes overall positive and we the importance of no difference between spinal and top-up EDAThe manuscript titled “The decision to delivery interval in emergency caesarean sections: Impact of anaesthetic technique and work shift” is a well written study with limitations due to the retrospective nature.I have three comments for possible revision: Please specify the top-up timing: The most interesting result as the similar onset of epidural top-up to spinal anesthesia without any given information about the used local anesthetic volume. The mixture and volumes used are addressed in the method section, we can’t provide single patient data about volume/time. When were top-ups injected? (in the delivery unit just following c-section decision or in OR?)This is explicitly addressed and added to discussion Please specify the type of spinal bupivacaine (hyper-/ iso-baric?) Hypebaric added The main limitation is the lack of details about cesarean delivery indications. It is not surprising that general anesthesia allowed to quickest possible operation start. But particularly general anesthesia group includes possibly more emergent cases leading to lower Apgar scores. It may give more insight to the reader, if the authors can give data about the number of neonates with pH<7.1 at birth. The aim was not to explicitly address neonatal outcome. We found that 14 out of the 64 neonates GA mothers had a Ph < 7.1, there was 2 neonates in spinal group of mothers (DDI 15 and 17 minutes), and only 1 out of the 41 mothers having top-up EDA (DDI 24 minutes). Added to the results." } ] }, { "id": "28440", "date": "30 Nov 2017", "name": "Tülay Özkan Seyhan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript titled “The decision to delivery interval in emergency caesarean sections: Impact of anaesthetic technique and work shift” is a well written study with limitations due to the retrospective nature. I have three comments for possible revision:\nPlease specify the top-up timing: The most interesting result as the similar onset of epidural top-up to spinal anesthesia without any given information about the used local anesthetic volume. When were top-ups injected? (in the delivery unit just following c-section decision or in OR?)\n\nPlease specify the type of spinal bupivacaine (hyper-/ iso-baric?)\n\nThe main limitation is the lack of details about cesarean delivery indications. It is not surprising that general anesthesia allowed to quickest possible operation start. But particularly general anesthesia group includes possibly more emergent cases leading to lower Apgar scores. It may give more insight to the reader, if the authors can give data about the number of neonates with pH<7.1 at birth.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-1977
https://f1000research.com/articles/6-1691/v1	15 Sep 17	{ "type": "Data Note", "title": "Draft genome of tule elk Cervus elaphus nannodes", "authors": [ "Jessica E. Mizzi", "Zachary T. Lounsberry", "C. Titus Brown", "Benjamin N. Sacks", "Jessica E. Mizzi", "Zachary T. Lounsberry", "C. Titus Brown" ], "abstract": "This paper presents the first draft genome of the tule elk (Cervus elaphus nannodes), a subspecies native to California that underwent an extreme genetic bottleneck in the late 1800s. The genome was generated from Illumina HiSeq 3000 whole genome sequencing of four individuals, resulting in the assembly of 2.395 billion base pairs (Gbp) over 602,862 contigs over 500 bp and N50 = 6,885 bp. This genome provides a resource to facilitate future genomic research on elk and other cervids.", "keywords": [ "Cervus elaphus nannodes", "genome draft", "mammalian genome assembly", "tule elk" ], "content": "Introduction\n\nTo date, the closest genomic resource for elk (Cervus elaphus) is a full mitochondrial assembly of white-tailed deer (Odocoileus virginianus), a distantly related cervid1. The present paper presents the first de novo genomic draft of the tule elk (C. elaphus nannodes). This California-endemic elk subspecies underwent a major genetic bottleneck when its numbers were reduced to as few as three individuals in the 1870s2,3. Although their numbers have increased to >5,000 today4, the historical bottleneck nevertheless left its mark on the elk’s genome, rendering it more homozygous than other elk subspecies.\n\nOur motivation for generating a genomic resource for the tule elk was to create a reference for identifying single nucleotide polymorphisms (SNPs) to develop assays to monitor elk population abundance and for related population genetic applications. Due to the relatively low coverage generated in this work (40X overall with an average of 10X coverage from each individual), we used the MEGAHIT metagenome assembler, which has been found to perform well on low-quality or low-coverage DNA sequencing in bacteria5.\n\n\nMethods\n\nElk were selected from four geographically distinct populations across northern California to maximize genomic diversity (San Luis Reservoir, California Valley, American Canyon, and the San Luis National Wildlife Refuge4). Genomic DNA was extracted from skin biopsies, which were obtained by the California Department of Fish and Wildlife as part of their elk management activities4. We extracted DNA from skin using Qiagen DNeasy blood & tissue kits (QIAGEN Inc., Valencia, CA), according to the manufacturer’s instructions. The DNA was then fragmented via sonication using a Bioruptor (Diagenode, Denville, NJ) to 300 to 400 base pairs (bp) prior to adapter ligation. After verification of fragment size range using agarose gel electrophoresis, NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Inc., Ipswich, MA) was used to ligate Illumina adapters. Multiplexed libraries were prepared using NEBNext Multiplex Oligos for Illumina (New England Biolabs) to individually barcode each of four individual elk. Barcodes were annealed using low-cycle polymerase chain reactions during library preparation. To assess library quality, trace analysis was performed using a Bioanalyzer 2100 (Agilent, Santa Clara, CA) and fluorometric DNA quantitation of libraries was performed using a Qubit fluorometer (Invitrogen, Carlsbad, CA) prior to equilibrating sample concentrations and pooling for sequencing. After library quality control, four samples (one from each population) were pooled in equimolar concentrations and submitted for paired-end sequencing. Samples were sequenced on an Illumina HiSeq 3000 at the DNA Technologies and Expression Analysis Core of the UC Davis Genome Center.\n\nSequencing quality on demultiplexed reads was evaluated using FastQC v0.11.3 (RRID:SCR_014583)6. The Illumina TruSeq3-PE sequencing adapters were removed using Trimmomatic v0.30 (RRID:SCR_011848)7 with the ILLUMINACLIP parameter set to TruSeq3-PE.fa:2:40:15. The TruSeq3-PE.fa sequence was downloaded from https://anonscm.debian.org/cgit/debian-med/trimmomatic.git/plain/adapters/TruSeq3-PE.fa. LEADING, TRAILING, and SLIDING parameters were set to 2, resulting in the removal of bases with a quality score of 2 or less according to a phred33 quality scoring matrix. The SLIDINGWINDOW parameter of 4:2 was used to clip reads once the quality score fell below 2 within the window. The MINLENGTH parameter set to 25 dropped any reads that fell below that length due to quality trimming. The demultiplexed, quality-filtered reads were interleaved using the interleave-reads.py script in khmer v2.0 (RRID:SCR_001156)8. The assembly was performed using MEGAHIT v1.0.59 on interleaved quality filtered reads. Genome statistical analysis was done using QUAST v3.0 (RRID:SCR_001228)10. All code used is publicly available at https://github.com/dib-lab/2017-tule-elk/.\n\n\nResults\n\nWe obtained 377,980,276 demultiplexed 150 bp paired-end raw reads, containing a total of 113.394 Gbp of sequence, or approximately 40X coverage of the approximately 3 Gbp tule elk genome. Sequence assembly resulted in the generation of a total genome sequence size of 2.395 Gbp. Reads were assembled into 602,862 contiguous sequences (\"contigs\") averaging 3,973 bp in length with a minimum contig length of 201 bp. The G+C content of the genome was 41.55%. The N50 was 6,885 bp and maximum contig length was 72,391 bp. Additional assembly statistics are available in Table 1. No contigs (e.g. under a certain size or likely to reflect repeats) were removed from the assembly.\n\nThis genome can serve as the basis for further genomic work on tule elk and other cervids, such as the development of a SNP assay to track elk population movement across increasingly developed northern Californian terrain. Furthermore, it is the first whole genome assembly available from the family Cervidae, providing a useful interim reference genome for bioinformatic analyses on other deer and elk species.\n\n\nData availability\n\nRaw reads are available in the SRA under the BioProject ID PRJNA345218. The genome draft is available at https://doi.org/10.6084/m9.figshare.5382565.v111.\n\nCode used in this study have been archived at http://doi.org/10.5281/zenodo.88793512", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nSupport for this project was provided by a grant to BNS from the California Department of Fish and Wildlife, FY1516 Big Game Management Program (Grant ID P1580009).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nJM would like to thank Luiz Irber, Camille Scott, and Lisa Johnson of the DIB lab at UC Davis for assistance with bioinformatics processing. We also thank C. Langner and J. Hobbs of the California Department of Fish and Wildlife for providing samples.\n\n\nReferences\n\nSeabury CM, Bhattarai EK, Taylor JF, et al.: Correction: Genome-Wide Polymorphism and Comparative Analyses in the White-Tailed Deer (Odocoileus Virginianus): A Model for Conservation Genomics. PLoS One. Public Library of Science. 2011; 6(2): e15811. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcCullough DR: The Tule Elk: Its History, Behavior, and Ecology. elibrary.ru. 1969. Reference Source\n\nSacks BN, Lounsberry ZT, Kalani T, et al.: Development and Characterization of 15 Polymorphic Dinucleotide Microsatellite Markers for Tule Elk Using HiSeq3000. J Hered. 2016; 107(7): 666–669. PubMed Abstract \| Publisher Full Text\n\nHobbs JH: Draft Conservation and Management Plan for Elk. Report to the California Department of Fish and Wildlife. 2014; 41.\n\nSeitz A, Nieselt K: Improving ancient DNA genome assembly. PeerJ. 2017; 5: e3126. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAndrews S: FastQC: a quality control tool for high throughput sequence data. 2010. Reference Source\n\nBolger AM, Lohse M, Usadel B: Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014; 30(15): 2114–20. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCrusoe MR, Alameldin HF, Awad S, et al.: The khmer software package: enabling efficient nucleotide sequence analysis [version 1; referees: 2 approved, 1 approved with reservations]. F1000Res. 2015; 4: 900. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLi D, Luo R, Liu CM, et al.: MEGAHIT v1.0: A fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods. 2016; 102: 3–11. PubMed Abstract \| Publisher Full Text\n\nGurevich A, Saveliev V, Vyahhi N, et al.: QUAST: quality assessment tool for genome assemblies. Bioinformatics. 2013; 29(8): 1072–75. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMizzi J, Lounsberry ZT, Brown CT, et al.: Tule Elk Draft Genome. figshare. 2017. Data Source\n\nMizzi J: dib-lab/2017-tule-elk: Ready for publication. Zenodo. 2017. Data Source" }	[ { "id": "26607", "date": "25 Oct 2017", "name": "Steve Olsen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes the generation of a draft genome (40X coverage from 4 animals) of the tule elk (Cervus elaphus nannodes). The research methods are fairly standard for the Illumina sequencing used. At 602,862 contigs, the genome is very prelminary and will require quite a bit of additional work in order for it to be applicable to a wide range of applications. The report basically falls into a category of a genome announcement.\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "3233", "date": "11 Dec 2017", "name": "Jessica Mizzi", "role": "Author Response", "response": "Thank you for your review of this paper." } ] }, { "id": "27606", "date": "16 Nov 2017", "name": "Rudiger Brauning", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors describe the generation of a draft assembly for tule elk in the style of a brief genome announcement. For SNP detection and primer design this assembly is fine. It could e.g. be used in combination with Genotyping by Sequencing on additional individuals.\nMaterials and methods are sound and provided in full.\nHowever a quick search of NCBI's taxonomy resource reveals that since June 2017 there is a genome assembly for red deer available https://www.ncbi.nlm.nih.gov/genome/10790. The authors therefore cannot claim to present the first whole genome assembly from the family Cervidae. Please change that statement.\nSuggested further improvements:\nResults\nI would have liked to see a figure for the total amount of sequence after filtering as a simple way of showing how good or bad the sequence run was.\nTable 1's readability would be improved by getting all figures to align right.\nI'd also recommend to add another assembly metric to look at the gene content; either using something like BUSCO or by mapping the refseq sequences of a related, well annotated species (e.g. cattle) against the draft genome.\nMethods\nSample collection and library prep I see that each individual has two tissue samples. The authors entered a sample ID into the 'tissue' field of NCBI's BioSample database. I'd recommend removing this and adding the animal ID in the 'isolate' field.\nPlease expand the entries in the 'isolation source' field. It says e.g. \"Am. Cyn\" which probably means American Canyon.\nBioinformatics processing Checking the code I believe the statement \"LEADING, TRAILING, and SLIDING parameters were set to 2\" should read \"LEADING and TRAILING parameters were set to 2\".\n\nIs the rationale for creating the dataset(s) clearly described? Yes\n\nAre the protocols appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and materials provided to allow replication by others? Yes\n\nAre the datasets clearly presented in a useable and accessible format? Yes", "responses": [ { "c_id": "3232", "date": "11 Dec 2017", "name": "Jessica Mizzi", "role": "Author Response", "response": "Thank you for your review of this paper. Version 2 has been edited to reflect the presence of the red deer genome and a citation to that genome has been made. Table 1 has been reformatted for readability. The changes you’ve requested to the NCBI BioSample entry have been made. The trimmomatic code in the Bioinformatics Processing section has been edited to remove the erroneous “SLIDING” parameter. We’ve added text to the first sentence of the results section that describes the quality of sequence data in terms of standard quality scores. We opted not to provide details on the gene content relative to a related genome as we felt this could be done more comprehensively in the future once the red deer genome has been published and peer-reviewed." } ] } ]	1	https://f1000research.com/articles/6-1691
https://f1000research.com/articles/5-1086/v1	02 Jun 16	{ "type": "Research Article", "title": "Towards understanding the molecular mechanism of cardiolipin transport in Salmonella typhimurium: interactions between an essential inner membrane protein YejM and its newly found ligand, YejL", "authors": [ "Uma Gabale", "Gene Qian", "Elaina Roach", "Susanne Ressl", "Uma Gabale", "Gene Qian", "Elaina Roach" ], "abstract": "Salmonella typhimurium is responsible for over 35% of all foodborne illness related hospitalizations in the United States. This Gram-negative bacterium possesses an inner and an outer membrane (OM), the latter allowing its survival and replication within host tissues. During infection, OM is remodeled by transport of glycerophospholipids across the periplasm and into the OM. Increased levels of cardiolipin in the OM were observed upon PhoPQ activation and led to the discovery of YejM; an inner membrane protein essential for cell growth involved in cardiolipin binding and transport to the OM. Another protein that might be playing a role in cardiolipin transport is YejL, as its gene is localized upstream of yejm on the same operon. Here we report how YejM was engineered to facilitate crystal growth and X-ray diffraction analysis. Furthermore, we present for the first time that YejL is a ligand for YejM. Successful structure determination of YejM and YejL will help us understand how they interact and how YejM facilitates cardiolipin transport to the OM. Ultimately, yejm, being an essential gene, may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium.", "keywords": [ "Salmonella typhimurium", "cell growth", "outer membrane", "membrane protein", "YejM", "YejL", "cardiolipin" ], "content": "Introduction\n\nSalmonella typhimurium is a Gram-negative bacterium responsible for over 35% of all foodborne illness-related hospitalizations in the United States (Painter et al., 2013). S. typhimurium possesses an additional outer membrane (OM) with an asymmetric lipid composition, that serves as a barrier to the environment allowing its survival and replication within host tissues (Dalebroux & Miller, 2014; Needham & Trent, 2013; Pagès et al., 2008). Mechanisms for the transport and assembly of OM lipopolysaccharides, proteins, and exopolysaccharides have been defined (Dong et al., 2006; Dong et al., 2014; Hagan et al., 2011; Whitfield & Trent, 2014); however, the transport of glycerophospholipids across the periplasm and insertion into the inner-leaflet of the OM is not well understood.\n\nInterestingly, increased levels of cardiolipin in S. typhimurium OM were observed upon PhoPQ regulator activation (Dalebroux et al., 2014). Recently the inner membrane protein YejM was shown to bind cardiolipin and be involved in OM formation (Dalebroux et al., 2015). Furthermore, YejM is known to be an essential gene in E. coli and was shown to be involved in intrinsic multidrug resistance (De Lay & Cronan, 2008; Duo et al., 2008). YejM is comprised of 586 amino acids forming five predicted N-terminal transmembrane helices, followed by an arginine-rich periplasmic random coil linker region, and a C-terminal periplasmic domain (Figure 1A), and it was shown that YejM associates as a tetramer in solution (Dalebroux et al., 2015). The arginine-rich linker region and periplasmic globular domain of YejM were shown to bind cardiolipin and are required for OM remodeling and cell growth (Dalebroux et al., 2015). The molecular mechanism of the interplay between PhoPQ system and YejM, and how cardiolipin molecules are transported to the OM, need further structural and functional investigation.\n\nA) Operon architecture containing genes yejl and yejm, YejM protein domain architecture, YejL structure (PDB ID: 2JRX) showing electrostatic surface pattern, black arrows indicating negative charged areas, and in cartoon representation the two intertwined dimer (chain A in green and chain B in blue) forming a four-helix bundle B) SDS-PAGE analysis of purified protein samples. C) Crystals of YejM241 under various conditions. D) Diffraction image of YejM241 crystal from condition C6.\n\nInterestingly the gene yejl is localized upstream of yejm on the same operon (Figure 1A), and encodes for the 8.5 kDa protein, YejL, that forms a homodimer (pdb ID: 2JRX) (Figure 1A). The function of YejL is not known; however based on the co-location on the operon and YejL’s negatively charged areas shown in the homodimer NMR structure, we hypothesize that YejL is a ligand to YejM. We further hypothesize that YejL binds to the same site on YejM as cardiolipin and therefore may be a regulator of YejM-mediated cardiolipin transport.\n\nHere we report how YejM was engineered to facilitate crystal growth and present successful crystallization conditions with preliminary X-ray diffraction analysis. We further report for the first time that YejL is a ligand of YejM. Future successful structure determination of YejM alone and in complex with YejL will help us to understand cardiolipin transport to the OM and may lead to new drug targets inhibiting the pathogenic properties of S. typhimurium.\n\n\nResults\n\nWe purified full-length YejM and periplasmic constructs YejM191-586 as described in (Dalebroux et al., 2015). The original construct YejM191-586 failed to crystallize and showed a degradation product after electrophoresis in the SDS polyacrylamide gel (Figure 1B, middle lane). To prevent degradation, reduce flexible protein parts and remove positively charged arginine clusters, we deleted the linker region A191 to E240 in the YejM191-586 construct, resulting in YejM241-586. These modifications were made to increase the chance of crystal growth. Initial crystals of YejM241-586 appeared after one week incubation at 18°C under different conditions; e.g. needle clusters in 2.8 M sodium acetate trihydrate pH 7.0, 0.1 M BIS-TRIS propane pH 7.0 (Hampton SaltRx condition A2, Hampton Research), needle clusters in 2.8 M sodium acetate (Hampton Index condition B12, Hampton Research), and rhombohedral crystals appeared in 3.5 M sodium formate pH 7.0 (Hampton Index HR conditions C1, Hampton Research). These early hit crystals diffracted poorly, only up to 6Å (data not shown) and optimization and up-scaling of the crystallization setup from a 96 well format to a 24 well format did not improve the diffraction quality. Further screening using (Hampton PEGRx HT screen, Hampton Research) resulted in new crystal forms grown in condition C6 (0.1 M HEPES pH 7.5, 12% w/v polyethylene glycol 3,350) and C4 (0.1 M Citric acid pH 3.5, 25% w/v polyethylene glycol 3,350). Screening around these two conditions led to crystals in a condition consisting of 0.1 M citric acid pH 4, 18% w/v polyethylene glycol 3,350 (Figure 1C). YejM241-586 crystals grown in condition C6 diffracted well, up to 1.6 Å (Figure 1D). Data indexing and scaling with XDS (Kabsch, 2010) and further analysis with AIMLESS (Evans, 2006) resulted in a data set up to 1.8Å resolution and good overall statistics (Table 1).\n\nThe gene yejl is localized upstream on the same operon as yejm. yejl encodes for a small (8.5 kDa) protein, YejL, that forms an intertwined four-helix bundle (PDB code: 2JRX) and has negatively charged regions located at the “tail” ends of the four-helix bundle (Figure 1A). The biological function of YejL is unknown, however, its presence on the same operon as YejM suggests that it may interact with YejM and function as a regulatory element. More specifically, we hypothesize that the negatively charged regions of YejL could be involved in specific interaction with the positively charged arginine-rich linker region of YejM. Notably, this arginine-rich linker region is proposed to bind cardiolipin and may be a crucial region for cardiolipin translocation by YejM (Dalebroux et al., 2015). To test our hypothesis that YejL is a ligand of YejM, we purified and mixed both proteins in different stoichiometric ratio and conducted Blue Native PAGE (BNE) and size-exclusion chromatography (SEC) for detection of population of higher molecular weight oligomers/complexes in the mixtures. Our SEC experiments showed that, increasing concentrations of YejL fraction into YejM191-586 or YejM241-586 fractions, respectively (Figure 2A) resulted in a shift in the apparent molecular weight that suggested formation of a higher molecular weight complex. In BNE, we observed a clear shift in position of YejL towards YejM191-586 and YejM241-586, which indicated binding between YejL and YejM191-586 or YejM241-586, respectively (Figure 2B). Our data do not show an apparent difference between YejL binding to YejM191-586 that includes the arginine-rich linker region, and YejM241-586 that lacks the linker region. Further analysis using isothermal titration calorimetry and/or microscale thermophoresis will be used to determine the exact binding affinity and potential changes thereof between the two YejM constructs and YejL.\n\nA) SEC chromatograms showing controls and mixtures of YejM191-586 with YejL (left) and YejM241-586 with YejL (right). B) BNE showing controls and mixtures of YejM191-586 with YejL (left) and YejM241-586 with YejL (right), BSA was used as size reference.\n\n\nConclusions\n\nHere we report successful crystallization using protein engineered specifically to enable crystal growth of YejM. Our initial X-ray data analysis of YejM241-586 crystals suggests a dimer assembly of the periplasmic domain. Therefore the membrane domain is very likely needed to form the YejM tetramer. We will use the current 1.8Å dataset (Table 1) for structure determination. We also aim to solve the co-crystal structure of YejL and/or cardiolipin bound to YejM. Ultimately these structures will help in understanding: i) where and how YejL and/or cardiolipin bind to YejM, ii) the role of YejL, iii) whether YejM’s architecture is that of a transporter or channel, and iv) the molecular mechanism of cardiolipin translocation to the OM of S. typhimurium.\n\n\nMaterial and methods\n\nInitial clones of full-length YejM 1-586 (YejM) (Uniprot ID P40709) in pBAD24 and the periplasmic domain of YejM 191-586 in pET28a plasmid are described in (Dalebroux et al., 2015). We used forward primer YejM241-586 5'-ccgcgcggcagccatatggctagcgcggtctccgttcagtacccg- 3' and reverse primer YejM241-586 5'-gcgggtactgaacggagaccgcgctagccatatggctgccgcgcgg- 3' to create a shorter construct of the periplasmic domain lacking the linker region resulting in YejM 241-586. Purification of YejM, YejM 191-586, and YejM 241-586 was performed as described previously (Dalebroux et al., 2015). Samples used for subsequent crystallization experiments were further purified by SEC using a Superose 6 increase 10/300 GL column (GE Healthcare) in buffer containing 50 mM Tris pH 8.0, and 150 mM NaCl. SEC buffer for YejM contained 20 mM HEPES, pH 7.5, 150 mM NaCl, and 0.02% Dodecyl-β-D-Maltopyranoside (DDM). The concentration of DDM was kept right above the critical micelle concentration throughout all subsequent experiments. YejM and YejM241-586 SEC peak fractions were pooled and concentrated with 30 kDa NMWL centricon (Millipore) to 12 mg/ml and up to 50 mg/ml, respectively. The purity of the samples was judged by polyacrylamide gel electrophoresis (Figure 1B).\n\nYejL construct (ID ER309-21.7), an E. coli homolog, was obtained from the Northeast Structural Genomics (NESG) consortium (http://www.nesg.org) with the vector ID: pET21_NESG. The pET21-YejL plasmid was transformed into E. coli BL21(DE3) (Novagen) competent cells. Overnight saturated cultures of transformed bacteria in Luria Bertani (LB) medium (EMD Chemicals Inc.) with 50µg/ml ampicillin (Dot Scientific Inc.) were grown at 37°C and diluted 1:200 in fresh Terrific Broth (TB) medium (Dot Scientific Inc.) with 50µg/ml ampicillin and further grown at 37°C at 200 rpm. The cultures were induced with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) (Teknova) when OD600 reached 0.5–0.8. The expression was carried out at 37°C for three hours. All further steps were performed at 4°C unless noted otherwise. Cells were harvested by centrifugation and resuspended in a buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 5 mM MgCl2. 10µg/ml DNAse (Sigma Aldrich), 1 mg/ml lyzosyme (Sigma Aldrich), protease inhibitor cocktail (Roche), and 0.1 mM Phenylmethane sulfonyl fluoride (PMSF) (Dot Scientific Inc.) were freshly added to the resuspended cells. Cells were lysed by passing several times through a microfluidizer (Divtech Equipment Company) and the cell debris was removed by centrifugation.\n\nThe clear cell lysate was bound to Ni-NTA resin (Qiagen) in batch mode and incubated on a rotarod at 4°C for 30 – 60 minutes. The slurry was loaded into a column and the flow through was collected under gravity. The column was washed first with 30 column volumes of Wash buffer 1 (25mM Tris pH, 8.0, 300mM NaCl and 25mM Imidazole); then with two column volumes of Wash buffer 2 (25mM Tris pH 7.5–8, 150mM NaCl, 75mM Imidazole). The bound protein was eluted from the column with elution buffer (25 mM Tris, pH 8.0, 150 mM NaCl, and 300 mM Imidazole) in 200–500µl increments. Elutions were monitored for protein content by Bradford test. YejL was further purified by SEC using a Superose 6 increase 10/300 GL column (GE Healthcare) equilibrated in a buffer containing 50 mM Tris, pH 8.0 and 150 mM NaCl. Peak fractions of YejL were consolidated and concentrated using 3 kDa NMWL centricon (Millipore).\n\nVapor diffusion crystallization of YejM 241-586 (5–50 mg/ml) was set up using 96-well crystallization plates (Hampton Research) with a Phoenix robot (ARI). Various sparse matrix screens were used to set up sitting drops with a drop sizes between 500 nl to 1 µl. Crystallization plates were incubated at 20°C and monitored for crystal growth in a MinstelTM HT crystal imaging and detection tower (Rigaku). Optimal crystal growth was obtained at a protein concentration of 4mg/ml.\n\n10 µM of YejM191-586 or YejM241-586 was mixed with varying concentrations (2.5, 5, 10, and 15 µM) of YejL separately. Samples were incubated at 4°C for minimum 1 hr, mixed with sample buffer (5% Coomassie Brillant Blue G250, 100mM Bis-Tris pH 7.5, 0.5M 6-aminocaproacid (Biorad) for a total individual sample volume of 30μL. The samples were loaded on Mini-protean TGX Any-kD precast gels (Biorad) and electrophoresis was carried out at 4°C at 100 V for 60–90 minutes. The gels were destained in a solution containing 5% ethanol and 7.5% acetic acid.\n\n10 µM of YejM191-586 and YejM241-586 were each mixed with varying concentrations (2.5, 5, 10, and 15 µM) of YejL separately. Each protein mixture was incubated overnight at 4°C and subsequently purified by size-exclusion chromatography using Superose 6 Increase 10/300 GL gel-filtration column (GE Healthcare) equilibrated with 50 mM Tris pH 8.0, 150 mM NaCl. The UV absorption profiles at 280 nm of each run were normalized and compared.\n\nCrystals were harvested using Litho loops (Molecular Dimensions) and Nylon loops (Hampton Research), submerged into paraffin and blotted until no phase separation was visible between paraffin and the excess crystallization solvent. Diffraction data of YejM241-586 crystals were collected at the Advanced Light Source beamline 4.2.2 in Berkeley CA at 100K, using an oscillation of 0.1–0.2° per image. Diffraction data were processed using iMosflm (Powell et al., 2013) or XDS (Kabsch, 2010) and scaled with Scala (Evans, 2006).\n\n\nData availability\n\nRaw diffraction data images were uploaded to the Coherent X-ray Imaging Data Bank (http://cxidb.org/id-42.html) and are available under CXIDB ID 42, DOI 10.11577/1252489 (Gabale et al., 2016).", "appendix": "Author contributions\n\n\n\nU.G. expressed and purified proteins, performed SEC and BNE, analyzed data and wrote manuscript, G.Q and E.R expressed, purified, crystallized and performed BNE, S.R. expressed, purified, crystallized, collected and analyzed data, designed experiments and wrote manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nWe thank: Prof. Samuel I. Miller and Dr. Zachary D. Dalebroux for constructs of full-length YejM and periplasmic domain of YejM191-586, Richard Pfuetzner for stimulating discussions, Jonathan T. Siler and Jennifer Wong for help with initial protein purification and crystallization experiments, Dr. Ardian Soca Wibowo for his excellent service at the macromolecular crystallization facility at Indiana University Bloomington, and Dr. Jay Nix for his excellent support at Molecular Biology Consortium Beamline 4.2.2 at the Advanced Light Source (Berkeley, CA).\n\n\nReferences\n\nDalebroux ZD, Edrozo MB, Pfuetzner RA, et al.: Delivery of cardiolipins to the Salmonella outer membrane is necessary for survival within host tissues and virulence. Cell Host Microbe. 2015; 17(4): 441–451. PubMed Abstract \| Publisher Full Text\n\nDalebroux ZD, Matamouros S, Whittington D, et al.: PhoPQ regulates acidic glycerophospholipid content of the Salmonella Typhimurium outer membrane. Proc Natl Acad Sci U S A. 2014; 111(5): 1963–1968. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDalebroux ZD, Miller SI: Salmonellae PhoPQ regulation of the outer membrane to resist innate immunity. Curr Opin Microbiol. 2014; 17: 106–113. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong C, Beis K, Nesper J, et al.: Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature. 2006; 444(7116): 226–229. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDong H, Xiang Q, Gu Y, et al.: Structural basis for outer membrane lipopolysaccharide insertion. Nature. 2014; 511(7507): 52–56. PubMed Abstract \| Publisher Full Text\n\nDuo M, Hou S, Ren D: Identifying Escherichia coli genes involved in intrinsic multidrug resistance. Appl Microbiol Biotechnol. 2008; 81(4): 731–741. PubMed Abstract \| Publisher Full Text\n\nEvans P: Scaling and assessment of data quality. Acta Crystallogr D Biol Crystallogr. 2006; 62(Pt 1): 72–82. PubMed Abstract \| Publisher Full Text\n\nGabale U, Qian G, Roach E, et al.: Dataset: Towards understanding the molecular mechanism of cardiolipin transport in Salmonella typhimurium: interactions between an essential inner membrane protein YejM and its newly found ligand, YejL. CXIDB. 2016. Data Source\n\nHagan CL, Silhavy TJ, Kahne D: β-Barrel membrane protein assembly by the Bam complex. Annu Rev Biochem. 2011; 80: 189–210. PubMed Abstract \| Publisher Full Text\n\nKabsch W: XDS. Acta Crystallogr D Biol Crystallogr. 2010; 66(Pt 2): 125–132. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Lay NR, Cronan JE: Genetic interaction between the Escherichia coli AcpT phosphopantetheinyl transferase and the YejM inner membrane protein. Genetics. 2008; 178(3): 1327–1337. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNeedham BD, Trent MS: Fortifying the barrier: the impact of lipid A remodelling on bacterial pathogenesis. Nat Rev Microbiol. 2013; 11(7): 467–481. PubMed Abstract \| Publisher Full Text\n\nPagès JM, James CE, Winterhalter M: The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat Rev Microbiol. 2008; 6(12): 893–903. PubMed Abstract \| Publisher Full Text\n\nPainter JA, Hoekstra RM, Ayers T, et al.: Attribution of foodborne illnesses, hospitalizations, and deaths to food commodities by using outbreak data, United States, 1998–2008. Emerg Infect Dis. 2013; 19(3): 407–415. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPowell HR, Johnson O, Leslie AG: Autoindexing diffraction images with iMosflm. Acta Crystallogr D Biol Crystallogr. 2013; 69(Pt 7): 1195–1203. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWhitfield C, Trent MS: Biosynthesis and export of bacterial lipopolysaccharides. Annu Rev Biochem. 2014; 83: 99–128. PubMed Abstract \| Publisher Full Text" }	[ { "id": "14132", "date": "03 Jun 2016", "name": "William T. Doerrler", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nHere, the authors report the expression, purification and isolation of diffractable crystals of the periplasmic domain of the essential salmonella protein YejM, putatively involved in transport of cardiolipin to the outer membrane. They also report interaction between the periplasmic domain of YejM and the small protein YejL produced from a gene immediately upstream and likely cotranscribed with YejM.\nYejM was identified by a screen for mutants defective in PhoPQ mediated outer membrane remodeling and was renamed PbgA (PhoPQ barrier gene A). The regions of the protein and their proposed functions are as follows: 1-190, transmembrane domain of the inner membrane; 191-240, arginine-rich linker that binds to cardiolipin and 240-586, the periplasmic domain seemingly required for interaction with OM. The authors report isolation of crystals of the YejM241-596 periplasmic domain and interaction of YejL with YejM241-586 and YejM191-586 truncated mutants. Sadly, the inability to crystalize YejM191-586 will forestall attempts to examine cardiolipin-YejM interactions.\nThis work represents a preliminary attempt to understand at the molecular level an extremely interesting process required for Salmonella virulence namely OM lipid remodeling. YejM/PbgA represents a novel virulence factor and potential drug target. The importance of its identification cannot be understated.\nMajor issues:\nThis reviewer questions the physiological relevance of the interaction between YejL and the YejM periplasmic domains. YejL is a 75 amino acid protein encoded by the gene immediately upstream of YejM. It is not predicted to contain a secretion signal and the PDB itself predicts it to be a soluble cytoplasmic protein. It is difficult to envision how it would interact with the periplasmic domain of YejM in a living cell. Cellular fractionation studies showing a periplasmic localization of YejL would need to accompany the data in Figure 2 in order for it to be meaningful. Genetic studies may also be useful- for example, does the YejL mutant show alterations in PhoPQ mediated survival or YejM tetramer formation?\n\nThe BNE gels are not convincing. Why does YejL migrate at such a large molecular weight? YejM migrates as a smear in both gels when loaded by itself. Adding YejL reduces the smear but it cannot be said that YejL alters the MW of YejM in either gel.\n\nMinor issues:\nWhy not call YejM by its new name PbgA throughout? It would cause less confusion since the senior author was associated with the publication reporting the discovery last year.\n\nFigure 1A, the membrane domain in the figure should be labeled 0-190 not 191-240. Also the “j” in “YejL structure” is partially obscured.\n\nI take issue with describing YejL as a ‘ligand”. To me and most biochemists a ligand is a small molecule that interacts with a target protein (receptor) causing some kind of conformational change and usually generation of a signal of some sort. What they are describing here is a protein-protein interaction and should be referred to as such, i.e. “YejL interacts with the periplasmic domain of YejM”.", "responses": [ { "c_id": "3213", "date": "11 Dec 2017", "name": "Susanne Ressl", "role": "Author Response", "response": "Major issues:1.) We performed cellular fractionation studies based on the reviewer's suggeestions and present the results in the supplemental section in the updated version of the manuscript. The suggested genetic studies are beyond our laboratory’s main focus and scope of revised manuscript; however these studies are being currently performed in the laboratory of Dr. Dalebroux (personal communication).2.) We improved the quality of our BNE experiments and show the results in the supplemental section of the updated version of the manuscript.Minor issues:1.) PbgA is not yet officially listed as an alternative name for YejM. Uniprot lists entries for PbgA as proteins that are Phospho-beta-glucosidases. To prevent confusion, we chose to use the official Uniprot database nomenclature of YejM (http://www.uniprot.org/uniprot/P40709).2.) This figure is not included in the updated version of the manuscript.3.) We agree that this was not the correct term to use and would rather term YejL as potential interaction partner of YejM.We thank both reviewers for their helpful critique." } ] }, { "id": "14811", "date": "06 Jul 2016", "name": "Yihua Huang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript, Gabele et al. reported the structure of YejL and preliminary X-ray crystallographic characterization of the periplasmic domain (Residues 241-586) of YejM, an inner membrane protein potentially responsible for transport of glycerophospholipid cardiolipin. The authors also tested interaction between YejL and YejM using blue native gel electrophoresis and size exclusion chromatography. In general, it is an interesting study, yet the following issues need to be addressed before accepted for indexation:\n\nIt is not proper to claim that YejL is a ligand of YejM. “Ligand” is dedicated for “receptors” only. Also, there is no strong genetic evidence to show that YejL is involved in cardiolipin transport.\n\nThe authors claimed that YejM is a dimer based on its periplasmic domain, and probably, also because of YejL forming a dimer. This is not clear as the authors did not study the oligomerization state of the full-length YejM . Other than this, when running SEC on superpose 6, the authors did not include a standard marker for reference. It ishard to judge if the periplasmic domain of YejM forms a dimer in solution.\n\nThe author believed that the negative charge surfaces of YejL might bind with the arginine-rich loop of YejM. Why the fragment of YejM (241-586) that seems to not include the arginine-rich loop still binds YejL ? Please explain.", "responses": [ { "c_id": "3212", "date": "11 Dec 2017", "name": "Susanne Ressl", "role": "Author Response", "response": "1.) We apologize for the incorrect use of terminology and would rather term YejL as potential interaction partner of YejM.2.) We carefully checked within the manuscript what section/sentence could have caused this reviewer’s understanding that we claimed YejM to be a dimer. We found the following sentence in the “Conclusions” section of our originally submitted manuscript: “Our initial X-ray data analysis of YejM241-586 crystals suggests a dimer assembly of the periplasmic domain, therefore the membrane domain is very likely needed to form the YejM tetramer”. In this sentence, we suggested the existence of a periplasmic domain YejM241-586 dimer based on our analysis of the crystallographic unit. In this analysis the content of the asymmetric unit that builds the crystal resulted in a dimer of YejM 241-586, and therefore we proposed it to be the likely physiologically relevant state of the periplasmic domain alone. From the SEC-MALS studies in (Dalebroux et al. 2015) that full-length YejM forms a tetramer when purified with the detergent DDM. Therefore we reasoned in the above stated sentence that further oligomerization into a tetramer must be facilitated by the trans-membrane domain. Whether is exists as a dimer or a monomer either in the native membrane, in the presence of specific lipids or purified using a different detergent is a matter of current investigation by other laboratories (Miller and Dalebroux labs).3.) Indeed, the arginine-rich linker region of YejM would be the most obvious binding region for YejL. However, we cannot exclude the possibility that YejL binds to solvent-accessible positively charged residues on the YejM241-586 surface." } ] } ]	1	https://f1000research.com/articles/5-1086
https://f1000research.com/articles/6-2119/v1	11 Dec 17	{ "type": "Opinion Article", "title": "Health Technology Assessment capacity development in low- and middle-income countries: Experiences from the international units of HITAP and NICE", "authors": [ "Sripen Tantivess", "Kalipso Chalkidou", "Nattha Tritasavit", "Yot Teerawattananon", "Kalipso Chalkidou", "Nattha Tritasavit", "Yot Teerawattananon" ], "abstract": "Health Technology Assessment (HTA) is policy research that aims to inform priority setting and resource allocation. HTA is increasingly recognized as a useful policy tool in low- and middle-income countries (LMICs), where there is a substantial need for evidence to guide Universal Health Coverage policies, such as benefit coverage, quality improvement interventions and quality standards, all of which aim at improving the efficiency and equity of the healthcare system. The Health Intervention and Technology Assessment Program (HITAP), Thailand, and the National Institute for Health and Care Excellence (NICE), UK, are national HTA organizations providing technical support to governments in LMICs to build up their priority setting capacity. This paper draws lessons from their capacity building programs in India, Colombia, Myanmar, the Philippines, and Vietnam. Such experiences suggest that it is not only technical capacity, for example analytical techniques for conducting economic evaluation, but also management, coordination and communication capacity that support the generation and use of HTA evidence in the respective settings. The learned lessons may help guide the development of HTA capacity in other LMICs.", "keywords": [ "priority setting", "health technology assessment", "health policy", "capacity building", "low- and middle-income countries" ], "content": "Introduction\n\nHealth technology assessment (HTA) has been widely recognized as a policy tool, which provides helpful information for allocating finite resources and ensures equitable access to needed technologies in the context of universal health coverage (UHC)1. HTA determines effects and implications of a variety of technology, not only medicines, vaccines, medical devices and procedures, but also social interventions whose aim is to improve health2. HTA involves research in multiple disciplines in order to assess cost effectiveness, budget impact, programmatic feasibility and social and ethical issues of health interventions3. The demand for HTA capacity in low- and middle-income countries (LMICs) is likely to grow, partly due to the adoption of the World Health Organization (WHO) regional committees’ and World Health Assembly resolutions during 2012 to 20144–6.\n\nHTA and its policy utilization is well established in high-income countries, such as Australia, Canada, and European countries7. On the contrary, HTA capacity in most LMICs are lacking, especially in making links between evidence and policy8–10. In many settings where UHC has been adopted as a national policy, policymakers express their concerns about the financial sustainability of the healthcare services and also the demand for priority setting tools11. However, local research capacity is inadequate to supply HTA evidence12, and the connection between research and policy is impeded by several factors, such as the lack of awareness, understanding, knowledge and adequate will on the part of policymakers, technical officers and researchers on HTA and its role in evidence-based decisions.\n\nDuring the past decade, capacity building programs for HTA have been initiated by international and regional organizations and networks. These programs usually involve short-course trainings for academics and policy analysts, convening of annual conferences, and development and distribution of HTA guides, such as methodological guidance13–15. Furthermore, regional networks mostly active in Asia (HTAsiaLink) and Latin America (RedETSA, PAHO and IADB) have helped share experiences on HTA between countriesa. In late 2000s, the UK’s National Institute for Health and Care Excellence (NICE) International and Thailand’s Health Intervention and Technology Assessment Program (HITAP) and their partner organizations agreed to establish partnerships to strengthen priority setting in LMICs in different regions.\n\nWhile health priority setting is indispensable, lessons drawing on the experiences of NICE and HITAP will be helpful for introduction of HTA at country level. This paper reviews the efforts of the two institutes to encourage evidence generation and utilization of research in decision making in five LMICs. It also discusses the system context, including supportive factors of and challenges in the capacity building mission for each study setting.\n\n\nIntroduction to NICE and HITAP, and their international units\n\nThe UK’s National Institute for Health and Clinical Excellence (NICE) was set up in 1999, an integral part of the government’s aim to introduce clinical governance, and improve the quality, equity and efficiency of a chronically underfunded National Health Service (NHS)16. In addition to a commitment to using scientific evidence of comparative clinical and cost effectiveness, NICE built up its core decision making processes based on the principles of multistakeholder engagement, transparency, conflict of interest management and contestability. Over the years its remit grew to include deciding on coverage of all new drugs introduced in the NHS, setting quality standards for all core services, including safe staffing levels, and issuing guidance on public health and social care.\n\nIn the UK, the role of independent institutions such as NICE in translating evidence into policy (‘knowledge brokers’) has been highlighted beforeb as has the application of this model of institutional capacity well beyond health, in social policy and practice, through the country’s What Works Evidence Centersc.\n\nAs NICE’s international reputation grew so did requests from overseas policy makers for support in strengthening their own systems and processes for making difficult decisions on how best to allocate their limited budgets. In response, NICE International was set up in 2008 to offer: “Advice on building capacity for assessing and interpreting evidence to inform health policy and on designing and using methods and processes to apply this capacity” for better health around the world through effective and equitable use of resources.\n\nEstablished in 2007, HITAP is a semi-autonomous research arm of Thailand’s Ministry of Health (MOH). Its main mission is to conduct HTA and provide evidence and recommendations to decision makers17. HITAP’s research, including cost-effectiveness studies and budget impact analysis, has been formally embedded as part of coverage decisions, i.e. development of reimbursable medicines list and benefit package of the Universal Health Coverage scheme (a government-financed health scheme for 75% of population). During the past eight years, over 150 studies were conducted in this institute; most of them have been fed into national policymaking process18,19. Besides the policy analysis role, HITAP implements strategies to support HTA introduction in the country, such as capacity building for HTA researchers and users; development of guidelines, tools and a database of studies; and knowledge management strategies. In 2013, HITAP International Unit (HIU) was established to coordinate research, capacity building, research dissemination, networking and other activities at regional and international levels.\n\nAs of 2015, NICE International works in 7 countries in Europe, Latin America, Africa, and Asia, while HIU works in 7 countries in Asia. The two institutes apply similar principles of selecting countries with which they work. First, the LMIC should have a goal to move toward UHC and a policy demand for HTA capacity building; second, government or not-for-profit organizations assigned as a focal point committed to absorb capacity building; third, local partners agree to work on policy-relevant case studies or pilot projects in a participatory, and transparent manner. Furthermore, NICE International and HITAP give a higher priority to countries that will enable their staff to learn more on technical and policy issues.\n\n\nConceptual framework for HTA capacity development in LMICs\n\nHTA capacity building programs in LMICs introduced by HITAP and NICE International have the primary goal to institutionalize policy research as a means for achieving the efficient use of the limited resources in the UHC context. The two organizations developed theory of change as their framework to guide strategy and activities (Figure 1). In this framework, equitable access to essential health care is an important long term aim of the capacity development effort. As suggested by the WHO, “… institutionalizing meant promoting structures and processes suitable to produce technology assessments that will be powerful in guiding policy and clinical practice towards the best possible health and cost outcomes”20. This indicates that HTA processes, which include topic selection, evidence generation, appraisal, and appeal in a participatory and transparent manner, involve technical and political aspects of decision making, and therefore, the need for capacity development in both these dimensions.\n\nBuilding technical capacity is relatively straightforward, as essential skills and knowledge for determining the impact of health technology can be developed through training of individual researchers. However, technical capacity for HTA practically relies on infrastructure and resources, such as data availability and management, expertise in related disciplines and collaborations, procedural guidelines to ensure research quality and protection from vested interests, and research grants, all of which are limited in LMICs12,21. Across the political dimension, HTA is not merely research but also a policy tool, since it generates evidence to inform policy decisions and practice. Institutionalizing HTA requires the capacity to connect the research community with the complex policymaking sphere a difficult task in resource-poor countries22 and even in developed economies23.\n\nIn target LMICs, the capacity building programs provide training, advising and mentoring staff to transfer expertise and knowledge on multidisciplinary research and policy development from HITAP, NICE International and their network organizations. Other activities include convening stakeholder-participatory processes of technology assessment and mobilization of financial resources, materials, expertise and information to support HTA introduction in target countries. The deliverables include HTA policy statements, road maps, government own resources committed, method and process guides, demonstration projects, case studies and trained technical officers, researchers and policy makers. In the next step of capacity development, it is expected that HTA-informed policy will be expanded and regularly introduced, as long as the three conducive elements of political commitment, stakeholder engagement and scientific integrity can be sustained.\n\n\nHITAP and NICE experiences\n\nThis paper illustrates key features of five programs that were purposively selected as case studies that capture different levels of priority setting capacity and achievement of the programs so far.\n\nFinanced by Global Alliance for Vaccine and Immunization (GAVI)’s Health System Strengthening program, a voucher scheme to improve maternal and child health (MCH) has been introduced in Myanmar since 2010. HITAP was invited by the WHO to provide technical support to the MOH to formulate financing strategy to address priority health problems24. Through a consultation process, the use of vouchers as financing approach to facilitate access to MCH services, provided by skilled birth attendants, was agreed upon by decision makers and partner organizations. A series of operational research was conducted in 2010 and 2011 to inform the scheme design and implementation, such as target population, voucher distributors, benefit package, value of vouchers, payment mechanisms and communication guidelines. Besides, cost-effectiveness analysis played a crucial role in the policy adoption, since its findings suggested that the scheme would offer value for money in saving lives of mothers and newborns25. Since May 2013, a pilot scheme has been introduced in one township26.\n\nThe capacity development in Myanmar did not aim to institutionalize HTA, but strengthening evidence-based policymaking capacity through the engagement of decision makers and key stakeholders in every step of the operational study, as well as conventional training on health systems research, economic analysis, and public communication. It was expected that in long term such an experience would have spill-over effects on other policy issues25. However, for such spill-over effects to be achieved and sustained, development partners who drive the majority of health spending in the country, would have to show political commitment to using evidence and due process in priority setting and to offer continual technical and financial support. Myanmar illustrates the important role of external partners in cultivating evidence-informed decision making in low income settings.\n\nHTA has been introduced in the Philippines since 1999 to inform coverage decisions for medicines, medical devices and procedures under PhilHealth, the national insurance scheme27. In 2013, as the Department of Health (DOH) adopted economic evaluation as a tool for the development of the national formulary28, it sought technical support from HITAP.\n\nWith grant from the Rockefeller Foundation, assessments of Pneumococcal conjugate virus (PCV) and Human Papillomavirus (HPV) vaccines for the national EPI were selected as demonstration studies. Aiming at transferring HITAP’s experience on vaccine assessment, Thai modelers provided guidance, access to existing economic evaluation models, and supervision to Filipino researchers. The studies suggested that both vaccines were cost effective, but countrywide HPV immunization program might be unaffordable29. The DOH decided to scale up PCV vaccine coverage, while requested reanalysis of HPV vaccination provided on two-dose schedule as opposed to the three-dose schedule, as applied in the economic evaluation, since a significant decrease in budget impact was expected.\n\nIt was considered that merely enhancing the technical aspect of decision making would be inadequate. Therefore, key players in immunization policy, including those in the DOH, national EPI and industry sector, were invited to discussion sessions on preliminary and final research findings. These aimed to fine tune the parameters and assumptions employed in the analysis, and also get policy stakeholders acquainted with HTA principles and processes. In parallel, NICE International and its partners, such as the EuroQOL team, offered training in methods of evaluation and helped review the country’s methodological and process guides for conducting HTA. Finally, with HITAP and NICE support, the Philippines joined HTAsiaLink and participated in an HTA for tobacco control initiative funded by APEC, ARCH. Capacity building through networks such as HTAsiaLink and ARCH can complement the bilateral technical cooperation activities described earlier.\n\nContinuous support from international partners is indispensable for keeping the momentum of HTA introduction in this country. A key lesson learnt is that the development of HTA capacity can be catalyzed by beginning with a topic that the technical experts have already assessed, which is advantageous because the experts then have materials and relevant experiences to share to the local partners. This led to the completion of these projects in one year, compared to three years for the Thai studies, and two international publications in well-known journals29,30.\n\nIn 2012, NICE worked with Vietnam’s MoH on quality improvement in stroke management, with an emphasis on performance indicators that can improve patient care in a cost effective way. Through a multistakeholder process led by local clinicians, NICE and its NHS partners helped adapt the international evidence to the local setting and identify those simple and effective measures such as the introduction of stroke units and early patient mobilization. The quality standards derived from this process are now being rolled out in a major Hanoi public hospital with a view to further scaling up countrywide through a ministerial directive issued in 201331.\n\nHealth economics research has existed in Vietnam since 1990s; however, a review in 2014 suggested that economic evaluations were limited in terms of scope and number32. In addition, the use of cost-effectiveness evidence to inform resource allocation has not yet been formalized, owing to the lack of policy demand and link between researchers and policymakers. From 2013 HITAP and NICE International jointly convened a capacity building program for priority setting in this country, in order to address the request of the MOH, as it pursued UHC for the population. A situation analysis was conducted to gain understanding on the current mechanisms for resource allocation; the need and demand for HTA; technical capacity; and political context including key interests for connecting research with policy33. The inception phase also involved raising awareness of stakeholders on HTA as a tool for priority setting. In this regard, the Vietnamese Health System and Policy Institute (HSPI) was appointed by the Health Minister as the national HTA focal point to collaborate with key stakeholders to develop HTA framework, roadmap, strategy and guidelines.\n\nIn 2016 the MOH commissioned HITAP to provide support the revision of the Vietnamese benefits package which led to the reform of benefit package of high-cost medicines and medical devices under the Vietnam Social Security Scheme (VSS)34. This reform would save 3,335 billion VND (147 million US$) each year of VSS budget without minimising health outcomes by removing inappropriate use of high-cost medicines.\n\nThe capacity building model in Vietnam was designed to facilitate HTA institutionalization, by empowering local stakeholders. To do so, the main strategies in both policy and technical domains drew on HITAP’s and NICE’s experiences of conducting policy-oriented research in Thailand and other LMICs. Nevertheless, all responsible partners were well aware of the differences in health system context, including the political environment and available resources, and therefore, the need for adaptation of particular elements. Despite this, common principles of technical robustness, transparency, social accountability and policy relevance in establishing HTA institutes and of due process, such as systematic topic selection, conduction of research, dissemination of results and policy integration. These concepts were operationalized by ensuring political commitment, sense of ownership among local institutional partners, engagement of a broad range of stakeholders, and independent HTA processes.\n\nBringing all the different streams together, the value of this work lies in using analytical techniques such as HTA for technology evaluation and quality standard development for service improvement to inform prioritization decisions, in other words, decisions to invest pooled resources in alternative programs and technologies with a view to improving overall health in the context of UHC. Stronger capacity on HTA in Vietnam can help generate local evidence to support quality standard development that is relevant to the local context.\n\nAt the request of the Minister of Health of Colombia and with seed funding from the World Bank and DFID, and, later, through a dedicated multi-year program (2008–2013) supported by the Inter-American Development Bank, NICE International and other partners, such as IECS and national universities, engaged with the Colombian authorities to help institutionalise the process for deriving a basic package, with an emphasis on technologies. In the context of strong political commitment for reform coming from the very top of the government, one of the triggers for the specific project and the initial invitation to NICE by IDB was perhaps the challenge set to the government by the country’s courts to integrate the two benefits packages (the less generous subsidised package with the more generous contributory one) and the procedural, technical and process problems posed by this decision35.\n\nThrough a series of visits, exchanges and analyses, and with capacity mobilised from across Latin America, including Argentina’s Institute for Clinical effectiveness and Health Policy (IETS)36 and in-country IDB expertise, NICE International, IECS contributed to an institutional and organisational blueprint, including an operational business plan, which evolved into legislation establishing IETS, the Institute for Health Technology Evaluation.\n\nA parallel stream of technical work concentrated on comparative assessment of selection of Western (Oregon Medicaid; the Netherlands, UK, Australia) and Latin American (Chile, Costa Rica, Brazil, Uruguay) national systems, in consultation with the MOH officials. The analysis described listing as well as pricing and reimbursement processes across the selected countries, as well as actual listing and pricing decisions on the 20 pharmaceutical products (such as bevacizumab, insulin glargine and tacrolimus) most commonly requested through judicial challenge of insurer’s exceptional committees in Colombia.\n\nThe capacity building stream focused on building technical skills within Colombian universities and professional organisations in HTA and clinical guideline development, through training courses, hosting Colombian delegations of policy makers and researchers in the UK, and secondment opportunities for Colombian colleagues – the first director of IETS spent 6 months working at NICE International as part of his post graduate degree at the London School of Hygiene and Tropical Medicine (LSHTM).\n\nThe political momentum for such an effort came from the President himself, as described in the Presidential blog, which also quotes former British PM, Tony Blair identifying NICE as one of his most important achievements for the UK’s healthcared. It has been a tough process. Over two years after its formal launch, IETS is positioning itself in an ever changing system faced with threats to its financial sustainability. Coordinating cross government functions is a challenge for IETS, both in terms of responding in a timely fashion to policy priorities and in terms of linking its analyses to existing decision making processes currently led by different directorates across the major healthcare stakeholderse. Perhaps the most important challenge, however, is the role of the courts, whose rulings on an individual basis undermine the very principle of UHC and invalidate any efforts by government to institutionalise priority setting. A recent ruling challenging the regulatory processf and a decision to mandate government to pay for an experimental treatment for a patient in a specialist centre in the USg, are two examples of the challenge Colombia is facing.\n\nHTA is very much about due process, with an emphasis on stakeholder engagement and participation. Kerala, a southern Indian State of 30 million people, has one of the lowest rates of maternal mortality in India. Nevertheless, with a maternal mortality rate of approximately 88 and every single death of a mother giving birth triggering wider social unrest and severe questioning of the authorities in the media37, reducing maternal mortality was deemed to be the State’s first priority. With a committed Principal Secretary and support by DFID, Rockefeller’s Joint Learning Network38 and the Wellcome Trust, NICE International worked with the State government and leading state professionals to develop and test out evidence-informed Quality Standards for tackling the top causes of maternal mortality, starting with post-partum haemorrhage (PPH)39. As in the case of Vietnam, the emphasis was on turning evidence-informed guidelines into locally relevant measurable performance indicators, through a consultative and transparent process. This was followed by assessing how specific measures could be implemented locally, taking account of costs and feasibility issues. This led to training programmes targeting all labour room staff, the development and roll out of a new maternity register and the purchasing and distribution of new disposable delivery kits, including simple equipment for measuring blood loss.\n\nScale up of the standards across both public and private hospitals throughout the State begun in 2013 and early results suggest a reduction in the incidence of PPH and in mortality rates in the pilot hospitals40, though it is still too early to draw final conclusions. In fact, even though the project introduced a new registry for measuring processes of care and outcomes, data availability, in particular baseline data needed in order to establish impact, proved to be an area in need of further strengthening.\n\nThe Kerala experience highlights the importance of involving healthcare professionals from the local constituency right from the beginning of any engagement. The success of the Kerala work relied heavily on the leadership of senior professionals from the Kerala Federation of OBGs, who had been leading for years on the only State-wide confidential enquiry into maternal deaths in India: “Why Mothers Die”39. It was this enquiry that helped identify cost-effective measures targeting the main causes of maternal death. A proactive press following the story from the very beginning, also played an important roleh.\n\n\nDiscussion\n\nThe five case studies illustrate the features of capacity building efforts for priority setting in different health system contexts. In the beginning, a situation analysis was conducted in each setting to determine political commitment, demand for priority setting, technical expertise in local institutes, as well as stakeholders’ attitudes and positions towards the introduction of formal priority setting mechanisms. Long-term availability of government budget to match with international grants was also explored, as local resources are necessary for sustainable evidence-based priority setting. Therefore, in some countries with notable constraints of resources, like Myanmar, the goal of capacity development was not HTA institutionalization, but rather to expose decision makers and technical officers with evidence-informed policymaking concept and practice.\n\nAlthough the situation analysis could help identify potentially-successful countries and strategies for enhancing HTA capacity, the dynamicity in the political context and its effects on the practice of evidence-informed policy decisions seems to be unavoidable, such as in the case of Colombia. Furthermore, delays in the conduct of capacity building activities were observed in some study settings, owing to inadequate management ability of focal-point institutes. This indicates the need for monitoring and evaluation, as well as risk assessment and risk management approaches as integral components of program implementation.\n\nThe five study settings have reached different stages of HTA institutionalization. In most countries the capacity building activities delivered observable outputs, according to the conceptual framework, such as trained personnel and demonstration research projects (Figure 1). However, the outputs obtained from program activities are inadequate for institutionalizing HTA41. The most challenging task for HITAP and NICE International is to foster political commitment, stakeholder participation and technical integrity of HTA research as key factors of intermediary and long-term outcomes, i.e. sustainable use of evidence in policymaking and institutionalized HTA, respectively. Furthermore, although HTA institutes have been operating in some countries like Colombia, the capacity to ensure their performance and achievements has to continually be strengthened and the broader political commitment to supporting them, at the highest level, maintained. Through the implementation of WHA resolution 67.23, LMICs should benefit from programs and activities under the auspice of the WHO and other international partners along the lines described above.\n\nLiterature on HTA capacity building in resource-poor countries suggests that political will, involvement of stakeholders, technical and financial support from international partners are crucial42–44. The capacity development programs managed by HITAP and NICE International contribute to the literature as such experiences help identify common success factors in the five study settings as follows:\n\n1) The concept of HTA needs to be endorsed by senior decision makers including not only politicians but also health officers; this can be achieved building on the reputation of local as well as international partners. The importance of political buy-in at the Presidential level in Colombia and Ministerial level in Vietnam are cases in point.\n\n2) The process of HTA capacity development should be demand-driven, based on local policy agenda in order to link HTA to policy decisions and also help build trust. In countries without demand for use of evidence in certain policy areas, long-term capacity development to encourage evidence-informed priority setting may not be worth the effort. In the Philippines, the government had a clear question (linked to vaccine listing) to be addressed. This demand triggered external support and also helped mobilize internal funds, leading to the country joining regional HTA networks and committing own resources to develop a designated HTA unit within DOH.\n\n3) It is important that local institutional partner(s) are capable of doing technical work and of convening other stakeholders in-country as part of the HTA process. Based on our experience, many academic institutions in LMICs do not interact with decision makers, which limit their ability to contribute to policy and HTA capacity building. Thus, MOH or an MOH designated institution are involved as major partners. In Vietnam, HSPI, working closely with MOH as the latter’s technical arm, has been a most valuable partner and a focal point, designated by the government, through which capacity of local universities has been harvested and applied to the task of HTA analyses.\n\n4) Ability of capacity building program managers to mobilize internal and external resources to support local partners is necessary. This is because health priority setting requires research in multiple disciplines which do not available in one single institute responsible for providing training courses and also for other facets of capacity building programs including management. Here the role of development partners in poorer countries such as Myanmar is critical. Without continuous support and coordination, evidence informed policy making can never become a reality relying on government efforts alone.\n\nThis paper offers a narrative of case studies over a short time period, making it hard to derive conclusions as to the success or failure of the capacity building model we are describing here. The information shared is in the form of direct experiences of the authors; as such, the information provided may not be neutral as it does not incorporate others’ viewpoints, for example, local partners and international organizations. However, these experiences have been published as scientific findings in peer-reviewed international journals.\n\nMoreover, it is too early to determine health outcomes of HTA-based policies in the five study countries, and this is not the main focus of our paper. Based on our experiences, this is a time consuming and deeply political process, and HTA processes are implemented sometimes after several years of HTA evidence generation. Owing to such limitations, this paper does not offer recommendations on effective models for health priority setting in LMICs, but sharing experiences of the two organizations and lessons learned in different contexts. Perhaps the most important component of our work is to empower local apply the technical and political process of making evidence-informed decisions.\n\nThe work on HTA capacity building within countries requires long-term effort and flexibility. As discussed earlier, it is difficult to plan well in advance the next steps, which is challenging to sustain such support in the long-term. For HITAP and NICE International, it has proven difficult to obtain funding for work that does not offer clear, measurable and certain deliverables. We are constantly faced with uncertainty and are subject to local champions and to political priorities guiding each country’s agenda.\n\nA further challenge more specific to low-income countries (LICs), has to do with capacity at regional level and the degree to which HTA processes can be regional as opposed to country specific. The European model of HTA, EUnetHTA, involving different levels of activities such as joint work on HTA, methodological guidelines, information technology tools, legal framework, and institutional structure13,14, may be applicable to regions such as APEC or the Americas. Societies such as INAHTA, HTAi and ISPOR can have a role to play in capacity building. Emerging models such as HTAsiaLink are also valuable means of strengthening capacity.\n\nLinked to the above to LICs, is the role of international donors in institutionalizing HTA as a technical and political tool to set priorities. The fact donors often control valuable resources for buying commodities (whereas MOH resources tend to be committed to infrastructure and salaries) and tend to have needed technical resources makes them the ideal conduit for HTA. However, their work concentrates on specific diseases (GF) and technologies (GAVI), making allocative efficiency considerations less applicable. With more countries interested in merging vertical programs with their own basic packages and with donor support declining, HTA is likely to become increasingly important.\n\nA final challenge is the role of the private sector in the process of building institutional, data and technical capacity for HTA. Within a transparent framework where interests can be managed on all sides and participation is encouraged, HTA can serve as an ideal platform for the healthcare products as well as the private insurance industry, reducing uncertainty regarding market access and helping set standards for managing providers, respectively. Our experience has been that HTA is an engagement tool that can benefit private players, many of whom still see it as a cost-containment measure.\n\n\nConclusion\n\nIntroducing evidence-informed priority setting in LMICs requires long-term, political commitment and support from politicians and senior health officers. Importantly, the direction for evidence generation and related capacity development should be shaped by local policy demand, which varies from setting to setting. Stakeholder and public participation in identifying HTA topics, conducting research and making policy decisions is a good practice. A key role of outsiders like the international units of NICE and HITAP is to provide general guidance on each step of HTA institutionalization that is relevant to conditions in particular countries. This also includes offering assistance for building technical, management and communication capacity of individuals and organizations that support the generation and use of HTA evidence in the respective settings.\n\n\nFootnotes\n\nahttp://programs.jointlearningnetwork.org/blog/2013/may/15/health-technology-assessment-useful-tool-countries-moving-toward-universal-health-c\n\nbhttp://www.lshtm.ac.uk/groups/griphealth/resources/grip_health_working_paper_3.pdf and references therein\n\nchttp://www.nice.org.uk/Media/Default/News/NestaAlliance_and_NICE_paper.pdf\n\ndSee here: http://wsp.presidencia.gov.co/Prensa/2010/Septiembre/Paginas/20100921_04.aspx\n\neSee for example: http://www.larepublica.co/economia/%C2%BFde-d%C3%B3nde-saldr%C3%A1-la-plata-para-cubrir-la-reforma-la-salud_41055 and here for a review of the broader challenges of the Colombian healthcare system: http://www.cgdev.org/blog/political-economy-uhc-colombia-version\n\nfConstitutional Court: Sentencia C-313/14. http://www.corteconstitucional.gov.co/comunicados/No.%2021%20comunicado%2029%20de%20mayo%20de%202014.pdf\n\nghttp://www.eltiempo.com/estilo-de-vida/salud/gobierno-apelara-medida-que-favorecio-trasplante-de-camila-abuabara/14802615\n\nhSee here: http://timesofindia.indiatimes.com/home/Kerala-health-department-has-decided-to-implement-the-quality-standard-for-maternal-care-in-8-maternity-hospitals-in-the-state-on-a-pilot-basis-this-year-This-will-be-followed-by-a-full-roll-out-to-all-maternity-hospitals-thereafter-said-Rajeev-Sadanandan-/articleshow/18036461.cms and here http://www.thehindu.com/todays-paper/tp-national/tp-kerala/clinical-guidelines-to-achieve-imr-reduction/article4916431.ece for early coverage.", "appendix": "Author contributions\n\n\n\nAll authors were involved in the design and implementation of the five capacity building programs discussed in this paper.\n\n\nCompeting interests\n\n\n\nAt the time of preparation of this manuscript, YT was program leader, and ST and NT were staff of HITAP. KC was director of NICE International.\n\n\nGrant information\n\nThis study was funded by the Thai Health-Global Link Initiative Project (TGLIP), the international Decision Support Initiative (iDSI, funded by the Bill & Melinda Gates Foundation and the Department for International Development, UK). The Health Intervention and Technology Assessment Program (HITAP) is funded by the Thailand Research Fund under the Senior Research Scholar Program on Health Technology Assessment (RTA5580010), the National Health Security Office, the Health Systems Research Institute and the Bureau of Health Policy and Strategy, Ministry of Public Health.\n\nThe findings, interpretations and conclusions expressed in this article do not necessarily reflect the views of the above funding agencies.\n\n\nReferences\n\nWHO: The World Health Report 2013: Research for Universal Health Coverage. Geneva. 2013. Reference Source\n\nINAHTA: HTA resources. 2010; [cited 2010 November 23]. Reference Source\n\nHailey D, Babidge W, Cameron A, et al.: HTA agencies and decision makers: An INAHTA guidance document. Stockholm: INAHTA. 2010. Reference Source\n\nWHO Regional Committee for Pan America: Health technology assessment and incorporation into health systems. (Resolution CSP28/11), Washington, DC. 2012. Reference Source\n\nWHO Regional Committee for Southeast Asia: Health intervention and technology assessment in support of universal health coverage. (Resolution SEA/RC66/R4), New Delhi. 2013. Reference Source\n\nWorld Health Assembly: Health intervention and technology assessment in support of universal health coverage. (Resolution WHA 67.23), Geneva. 2014. Reference Source\n\nWhyte P, Hall C: The Role of Health Technology Assessment in Medicine Pricing and Reimbursement. Pharmaceutical Pricing Policies and Interventions. Geneva: WHO. 2013. Reference Source\n\nSinger ME: Cost-effectiveness analysis: Developing nations left behind. Pharmacoeconomics. 2008; 26(5): 359–361. PubMed Abstract \| Publisher Full Text\n\nTarn Y, Hu S, Kamae I, et al.: Health-Care Systems and Pharmacoeconomic Research in Asia-Pacific Region. Value Health. 2008; 11 Suppl 1: S137–S155. PubMed Abstract \| Publisher Full Text\n\nOortwijn W, Mathijssen J, Banta D: The role of health technology assessment on pharmaceutical reimbursement in selected middle-income countries. Health Policy. 2010; 95(2–3): 174–184. PubMed Abstract \| Publisher Full Text\n\nWHO: Making fair choices on the path to universal health coverage. Final report of the WHO Consultative Group on Equity and Universal Health Coverage. Geneva. 2014. Reference Source\n\nGlassman A, Chalkidou K, Giedion U, et al.: Priority-Setting Institutions in Health: Recommendations from a Center for Global Development Working Group. Global Heart. 2012; 7(1): 13–34. PubMed Abstract \| Publisher Full Text\n\nEuropean Commission: HTA Network adopts its Strategy for EU cooperation on Health Technology Assessments. 2014; [cited 2014 29 November]. Reference Source\n\nKristensen F, Lampe K, Chase DL, et al.: Practical tools and methods for health technology assessment in Europe: Structures, methodologies, and tools developed by the European network for Health Technology Assessment, EUnetHTA. Int J Technol Assess Health Care. 2009; 25 Suppl 2: 1–8. PubMed Abstract \| Publisher Full Text\n\nHTAi: Health Technology Assessment International. 2014; [cited 2015 9 January]. Reference Source\n\nDepartment of Health: The New NHS: modern, dependable. 2007; [cited 2014 5 October]. Reference Source\n\nTantivess S, Teerawattananon Y, Mills A: Strengthening cost-effectiveness analysis in Thailand through the establishment of the Health Intervention and Technology Assessment Program. Pharmacoeconomics. 2009; 27(11): 931–945. PubMed Abstract \| Publisher Full Text\n\nTeerawattananon Y, Tritasavit N, Suchonwanich N, et al.: The use of economic evaluation for guiding the pharmaceutical reimbursement list in Thailand. Z Evid Fortbild Qual Gesundhwes. 2014; 108(7): 397–404. PubMed Abstract \| Publisher Full Text\n\nMohara A, Youngkong S, Velasco RP, et al.: Using health technology assessment for informing coverage decisions in Thailand. J Comp Eff Res. 2012; 1(2): 137–146. PubMed Abstract \| Publisher Full Text\n\nWHO: Institutionalization of Health Technology Assessment. Geneva. 2001. Reference Source\n\nIglesias CP, Drummond MF, Rovira J, et al.: Health-care decision-making processes in Latin America: problems and prospects for the use of economic evaluation. Int J Technol Assess Health Care. 2005; 21(1): 1–14. PubMed Abstract \| Publisher Full Text\n\nLiverani M, Hawkins B, Parkhurst JO: Political and Institutional Influences on the Use of Evidence in Public Health Policy. A Systematic Review. PLoS One. 2013; 8(10): e77404. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLehoux P, Blume S: Technology assessment and the sociopolitics of health technologies. J Health Polit Policy Law. 2000; 25(6): 1083–1120. PubMed Abstract \| Publisher Full Text\n\nHITAP: Feasibility study of the Community Health Initiative for Maternal and Child Health in Myanmar. Nonthaburi, 2010.\n\nTeerawattananon Y, Tantivess S, Werayingyong P, et al.: Evidence-informed policy formulation: the case of the voucher scheme for maternal and child health in Myanmar. WHO South East Asia J Public Health. 2014; 3(3): 285–288. PubMed Abstract \| Publisher Full Text\n\nWHO: Mid-term review of Maternal and Child Health Voucher Scheme. Yedarshey Township, Nay Pyi Taw, Yangon: WHO Country Office, Myanmar, 2014. Reference Source\n\nDe Rosas-Valera M: Health technology assessment in the Philippines. Int J Technol Assess Health Care. 2009; 25(Supp 1): 231–233. PubMed Abstract \| Publisher Full Text\n\nDe Rosas-Valera M: Health technology assessment in the Philippines. 2013; [cited 2015 9 January]. Reference Source\n\nGuerrero AM, Genuino AJ, Santillan M, et al.: A cost-utility analysis of cervical cancer screening and human papillomavirus vaccination in the Philippines. BMC Public Health. 2015; 15: 730. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHaasis MA, Ceria JA, Kulpeng W, et al.: Do Pneumococcal Conjugate Vaccines Represent Good Value for Money in a Lower-Middle Income Country? A Cost-Utility Analysis in the Philippines. PLoS One. 2015; 10(7): e0131156. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMedical Services Administration: According to the Decision number 4858/QD-BYT December 03, 2013 of Minister of Health, Vietnam. Hanoi: Ministry of Health, 2013.\n\nTran BX, Nong VM, Maher RM, et al.: A systematic review of scope and quality of health economic evaluation studies in Vietnam. PLoS One. 2014; 9(8): e103825. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHernandez-Villafuerte K, Li R, Towse A, et al.: International Decision Support Initiative (iDSI): Mapping of priority-setting in health in 17 low and middle countries across Asia, Latin America, and Africa. London: Office of Health Economics, 2015. Reference Source\n\nTeerawattananon Y, et al.: Chapter 17 More Than A List: Reforming a Country’s Health Benefit Package-- A Rigorous Approach to Tackle Costly Overutilization. In What’s In What’s Out: Designing Benefits for Universal Health Coverage. A Glassman, U Giedion, and P Smith, Editors, Brookings: Washington D.C., 2017.\n\nYamin A, Parra-Vera O: How do courts set health policy? The case of the Colombian Constitutional Court. PLoS Med. 2009; 6(2): e1000032. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNICE: NICE International Assists the Colombian Ministry of Social Protection. 2009; [cited 2014 2 December]. Reference Source\n\nRajagopal K: Where grieving mothers struggle to find answers. The Hindu, 2013; [cited 2014 29 November]. Reference Source\n\nPerabathina S: Developing quality standards for post-partum haemorrhage to reduce maternal mortality in Kerala. 2012; [cited 2014 29 November]. Reference Source\n\nPaily V, Ambujam K, Thomas B, eds.: Why Mothers Die Kerala 2006 – 2009. Second Report of Confidential Review of Maternal Deaths, Kerala 2006 to 2009. Kerala Federation of Obstetrics & Gynaecology: Kerala, 2012. Reference Source\n\nMaya C: Kerala’s MMR comes down to 66. 2014; [cited 2014 29 November]. Reference Source\n\nOortwijn W, Broos P, Vondeling H, et al.: Mapping of health technology assessment in selected countries. Int J Technol Assess Health Care. 2013; 29(4): 424–434. PubMed Abstract \| Publisher Full Text\n\nTeerawattananon Y, Tantivess S, Yothasamut J, et al.: Historical development of health technology assessment in Thailand. Int J Technol Assess Health Care. 2009; 25(Supplement 1): 241–252. PubMed Abstract \| Publisher Full Text\n\nKaló Z, Bodrogi J, Boncz I, et al.: Capacity Building for HTA Implementation in Middle-Income Countries: The Case of Hungary. Value Health Reg Issues. 2013; 2(2): 264–266. Publisher Full Text\n\nDoaee Sh, Olyaeemanesh A, Emami Sh, et al.: Development and implementation of health technology assessment: a policy study. Iran J Public Health. 2013; 42(Supple 1): 50–54. PubMed Abstract \| Free Full Text" }	[ { "id": "28885", "date": "21 Dec 2017", "name": "Kanchan Mukherjee", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article is a descriptive account of five case studies involving the work of NICE International and HITAP in LMICs. This being an opinion piece not all arguments are supported by evidence from literature. The Theory of Change (ToC) model is excellently conceived and can be a very useful tool for future analysis of HTA work in these countries. However, this paper would benefit from the following:\nThere needs to be updated information in places. eg. page 4, 2nd line, mentions country partners of NICE International and HITAP as of 2015. Considering the fact that this is end of 2017, an updated information would be better appreciated. Also, in page 5, in the section on Philippines, it would be good to update whether the HPV vaccination schedule was adapted as a two day/three day schedule. Currently, it reads incomplete. The limitations of this paper are well articulated especially the acknowledgement that other view points exists. The author’s mention in the limitations that “However, these experiences have been published as scientific findings in peer-reviewed international journals”. In this context, a list of references of these experiences/other view points (perhaps one/two from each country) can be included in references. There are some minor editing/typological errors which can be corrected.\nWith the above changes the paper can be indexed.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Partly\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [] }, { "id": "28888", "date": "28 Dec 2017", "name": "Justice Nonvignon", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAbstract “Such experiences suggest that it is not only technical capacity, for example analytical techniques for conducting economic evaluation, but also management, coordination and communication capacity that support the generation and use of HTA evidence in the respective settings\"\n\nThe above statement is not exactly clear from the country write ups. Perhaps authors could stress on each component (i.e. management, coordination and communication) in each case, by pointing out clearly how this happened for each case in the narrative. For example authors made mention of some delays in the conduct of capacity building activities in some settings due to inadequate management ability of focal point institutes. This is not clearly presented in the narrative. We suggest this be clearly outlined in the narrative for each case or for those that it is applicable before those conclusions are made in the abstract.\n\nMain text The objectives of the paper do not come out clearly. Since the focus of the paper is on HTA capacity building, it would be great to tease out for each case:\nWhat kind of trainings were given The baseline skills of staff trained e.g. for researchers, did they have any basic understanding of HTA? How these people were selected If they required to have some particular basic skills before they qualified to be trained\n\nFor each case study, readers would benefit from a highlight\nThe stage of HTA institutionalisation they were in The challenges of the capacity development project Supportive factors The type of stakeholders that were involved in the process Participation of stakeholders; their involvement and how that facilitated the process (this is done very well for the Philippines case study)\n\nFor the Colombian case, readers would benefit from a brief comment on the types of national health insurance that existed, how HTA is been used for each and its challenges like the numerous context in courts, highlighting on the reasons for the context, before this is introduced under the discussion section. As it stands now, readers will need priori knowledge of the Columbian health system and national health insurance scheme to understand and appreciate the context under which the discussion is made.\n\nDiscussion The paper could benefit from the following discussion points (that is if authors have such information)\nSince for each case study, each country was at a different stage of HTA and its use in decision making, it would be great to highlight on the skill set that was already available for countries such as the Philippines and Columbia, and the difference it made in terms of knowledge transfer Did the context under which HITAP and NICE went to develop capacity in each case affect the whole process of HTA institutionalization, including capacity development: in terms of acceptance, preparations for it, etc. Were the challenges, available skills, supportive factors and stakeholder participation differ from case to case especially in terms of the stage of HTA that they were in?\n\nAuthors need to review the text for some language errors. For instance, there are some typos under limitations section on page 8 and future challenges section on page 9 and some incomplete sentences.\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-2119
https://f1000research.com/articles/6-2113/v1	08 Dec 17	{ "type": "Software Tool Article", "title": "Tessellate & Montage: Molecular analytics of cyclic conformations", "authors": [ "Christopher Barnett", "Kevin Naidoo", "Kevin Naidoo" ], "abstract": "The conformations and shapes of macromolecular structures in biological and synthetic materials often define the macroscopic functions of the systems. Tessellate and Montage provide a standardized toolset for rapid reporting of large datasets allowing comparisons of cyclic molecule conformations (ring pucker) from structural databases and simulation trajectory data. This facilitates an understanding of the dynamic transition between common conformations and the flexible range in a ring that underlies molecular behaviour and recognition properties.", "keywords": [ "multiqc", "conformation", "saccharides", "visualisation", "macrocycles" ], "content": "Introduction\n\nCyclic molecules and systems consisting of cyclic molecules play central roles in several chemical and biological processes1,2. RNA, DNA, proteins and carbohydrates are polymers that all contain cyclic moieties that exhibit wide ranging conformational variety. For example, the puckering of fructose can be used to illustrate the value of determining conformational statistics as recognition and binding to Fructokinase is due to pucker specificity. Similarly different sugar puckers of ribose in RNA and de-oxyribose in A-DNA and Z-DNA account for the molecular shape of the respective nucleotide helices2. In DNA-protein complexes any kink in the DNA axis is accompanied by a change in deoxy-ribose pucker, demonstrating the important connection pucker plays in molecular structure and flexibility3,4. In enzymatic transition states for glycosyl hydrolases and transferases, conformations change to provide the correct electronic state for the reaction to take place5.\n\nConformational analyses, which recognise shapes (Figure 1), are important for finding transition state leads, understanding molecular recognition events, ligand fitting in macrocycles and for molecular pattern matching.\n\nWe created a standardized toolset called Tessellate & Montage for comparisons of cyclic molecule conformations (ring pucker) from structural databases and simulation trajectory data. Tessellate can readily find, calculate and quantify cyclic conformations of cycles and macrocycles in datasets. Montage forked from MultiQC6 is a transferable reporting package for visualizing the conformations of cycles. Montage addresses the need to compare multiple molecular data sets by incorporating results from multiple analyses into a single report. Similar tools for calculating ring conformations are shown in Table 1, however these tools do not provide methods for calculating the ring pucker of macrocycles, nor do they have the reporting capability for comparing multiple types of molecular data sets in a single report. Existing pucker models include perpendicular displacements from a mean plane7,8, intracyclic torsions9,10 and triangular tessellation11–15.\n\nA further aim was to ensure that these tools (Tessellate and Montage) function independently from informatics workflows systems, while also being readily incorporated and fully functional as part of systems such as Galaxy21 and the Glycome Analytics Platform22.\n\n\nMethods\n\nTessellate is a package for quantifying ring conformation and qualifying the conformer in terms of a canonical conformer (e.g. chair, boat). Developed in Python, Tessellate uses BioPython23 for accessing protein data bank files. Using Tessellate with Visual Molecular Dynamics (VMD) is possible through a tcl script provided. Visualising the results of this analysis is done using Montage.\n\nMontage is a fork of the excellent MultiQC6 and provides reporting templates and functionality for computational chemistry and chemical glycobiology. Previously we prototyped reporting within a web platform, the aim here was to ensure that the visual reports connected easily into workflow systems such as Galaxy21, GAP22 and were also transferable outside of these platforms.\n\nThese tools were designed to work in Linux.\n\nTriangular tessellation for ring sizes 5–8 – Implemented as per the following papers11,14,15. Protein and water residues in the input structures are ignored by default.\n\nNomenclature and ring ordering – Conformational qualification depends on a consistent ring ordering according to the IUPAC definition24. Residues atoms are matched to an internal ligand dictionary and aligned in best-possible cyclic order to prevent atom ordering mistakes.\n\nRing finding algorithm – Cycles are detected using the Smallest Set of Smallest Rings (SSSR) ring perception algorithm25.\n\nConformational quantitation – Coordinates are matched to known canonical conformers coordinates. The best match is returned.\n\nMacrocyclic – Macrocycle conformations are returned based on the centre of geometry of any subsidiary cycles.\n\nRequirements: Python 3, Linux. For PDB analysis, BioPDB is required. Detailed installation instructions, package requirements and usage examples are provided in the README file of the released software (see Software availability section).\n\nTessellate reads in molecular data and outputs a summary of cyclic conformations (Input file types: list containing PDB IDs, PDB file, list of atomic coordinates. Output file types: JSON, txt). Montage reads the summary from Tessellate and produces a html report with a summary chart and charts for 5,6,7,8 rings found in the inputs (Input file types: JSON, txt. Output file types: HTML).\n\nUsage example:\n\nThe workflow is as follows:\n\n1. Choose molecular data e.g. from the PDB or use VMD to create a list of atomic coordinates from molecular simulation data.\n\n2. Run Tessellate on the Linux command line e.g. `tessellate data/usecase-*DNA --input-format=pdblist --output-format=json --output-dir=output-usecase-rnadna`.\n\n3. Run Montage on the Linux command line e.g. `multiqc output-usecase-rnadna -m montage_tessellate`.\n\n4. Open report using a web browser.\n\nSimulation – Ribose in vacuo biased molecular dynamics calculation\n\nThe conformational changes during a biased free energy simulation for ribose. This analysis can be used to identify patterns in conformational change and confirm adequate sampling of phase space (Figure 2). VMD was used to open and read coordinate and trajectory files, and write out the atomic coordinates of the ribose 5 membered ring. The atomic coordinates are read in by Tessellate, producing pucker coordinates stored as JSON. MultiQC creates an HTML report from the JSON input.\n\nComparative conformational character of DNA and RNA\n\nThe nucleotides A-DNA and Z-DNA tend to be C3’-endo (3E), but in B-DNA the tendency is to C2’-endo (2E); this accounts for the different molecular shape of the respective nucleotide helices. To explore this, PDB structures that are representative of A, B and Z DNA (1ANA, 3BSE, and 2DCG) were analysed (see Operation section and Figure 3).\n\nCyclodextrins\n\nAn analysis of alpha cyclodextrin (ACD) from the PDB shows the glucose monomer conformations are mostly 4C1 (85.9%) with few deviations (Figure 4). The ring pucker of the macrocycle, as defined by treating the centre of geometry for each glucose in cyclodextrin as an atom, varies between planar (P) and half chair (H, e.g. 3H4, 5H4,2H3) conformations.\n\nResults from the three use cases can be found in Supplementary File 1.\n\n\nConclusions\n\nTessellate enables users to compile datasets from the reservoir of PDB and simulation produced data. From this ensemble of experimentally and computationally determined ring structures, the relation between cyclic conformational preference and other variables can be discovered using Montage.\n\nAbbreviations: ACD: Alpha Cyclodextrin; GAP: Glycome Analytics Platform; JSON: JavaScript Object Notation; PDB: Protein Data Bank; SSSR: Smallest Set of Smallest Rings\n\n\nSoftware availability\n\nLatest source code:\n\nTessellate: https://github.com/scientificomputing/Tessellate\n\nMontage: https://github.com/scientificomputing/Montage\n\nArchived source code as at the time of publication:\n\nTessellate: https://doi.org/10.5281/zenodo.106865626\n\nMontage: https://doi.org/10.5281/zenodo.106869227\n\nTessellate License: Apache 2.0\n\nMontage License: GNU GPLv3", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis publication is based on research that has been supported in part by the University of Cape Town’s Research Committee and the National Research Foundation of South Africa (NRF; grant 87956). This work is based in part upon research supported by the South African Research Chairs Initiative of the Department of Science and Technology and the NRF (grant 48103).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Zip file containing Tessellate and Montage outputs for the 3 use cases:\n\ntessellate_report_usecase-timeseries.json\n\ntessellate_report_usecase-ADNA.json\n\ntessellate_report_usecase-BDNA.json\n\ntessellate_report_usecase-ZDNA.json\n\ntessellate_report_usecase-ACD.json\n\nmultiqc_report_usecase1.html\n\nmultiqc_report_usecase2.html\n\nmultiqc_report_usecase3.html\n\nClick here to access the data.\n\nThe datasets are kept at https://github.com/scientificomputing/tessellate/tree/master/data (see Software availability section).\n\n\nReferences\n\nDwek RA: Glycobiology: Toward Understanding the Function of Sugars. Chem Rev. 1996; 96(2): 683–720. PubMed Abstract \| Publisher Full Text\n\nVarki A: Biological roles of oligosaccharides: all of the theories are correct. Glycobiology. 1993; 3(2): 97–130. PubMed Abstract \| Publisher Full Text\n\nSundaralingam M: CONFORMATIONS OF THE FURANOSE RING IN NUCLEIC ACIDS AND OTHER CARBOHYDRATE DERIVATIVES IN THE SOLID STATE. J Am Chem Soc. 1965; 87(3): 599–606. PubMed Abstract \| Publisher Full Text\n\nTolstorukov MY, Jernigan RL, Zhurkin VB: Protein-DNA hydrophobic recognition in the minor groove is facilitated by sugar switching. J Mol Biol. 2004; 337(1): 65–76. PubMed Abstract \| Publisher Full Text\n\nDavies GJ, Ducros VM, Varrot A, et al.: Mapping the conformational itinerary of beta-glycosidases by X-ray crystallography. Biochem Soc Trans. 2003; 31(Pt 3): 523–527. PubMed Abstract \| Publisher Full Text\n\nEwels P, Magnusson M, Lundin S, et al.: MultiQC: summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016; 32(19): 3047–8. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKilpatrick JE, Pitzer KS, Spitzer R: The Thermodynamics and Molecular Structure of Cyclopentane1. J Am Chem Soc. 1947; 69(10): 2483–2488. Publisher Full Text\n\nCremer D, Szabó KJ: Ab initio Studies of Six-Membered Rings, Present Status and Future Developments. In Conformational Behavior of Six-Membered Rings: Analysis, Dynamics, and Stereoelectronic Effects, Juaristi E, Ed. VCH Publishers: 1995; 59. Reference Source\n\nBérces A, Whitfield DM, Nukada T: Quantitative description of six-membered ring conformations following the IUPAC conformational nomenclature. Tetrahedron. 2001; 57(3): 477–491. Publisher Full Text\n\nGeise HJ, Altona C, Romers C: The relations between torsional and valency angles of cyclopentane. Tetrahedron Lett. 1967; 8(15): 1383–1386. Publisher Full Text\n\nHill AD, Reilly PJ: Puckering coordinates of monocyclic rings by triangular decomposition. J Chem Inf Model. 2007; 47(3): 1031–1035. PubMed Abstract \| Publisher Full Text\n\nBocian DF, Pickett HM, Rounds TC, et al.: Conformations of cycloheptane. J Am Chem Soc. 1975; 97(4): 687–695. Publisher Full Text\n\nJoshi NV, Rao VSR: Flexibility of the pyranose ring in α- and β-D-glucoses. Biopolymers. 1979; 18(12): 2993–3004. Publisher Full Text\n\nKhalili P, Barnett CB, Naidoo KJ: Interpreting medium ring canonical conformers by a triangular plane tessellation of the macrocycle. J Chem Phys. 2013; 138(18): 184110. PubMed Abstract \| Publisher Full Text\n\nBarnett CB, Naidoo KJ: Ring puckering: a metric for evaluating the accuracy of AM1, PM3, PM3CARB-1, and SCC-DFTB carbohydrate QM/MM simulations. J Phys Chem B. 2010; 114(51): 17142–54. PubMed Abstract \| Publisher Full Text\n\nCremer D: Calculation of puckered rings with analytical gradients. J Phys Chem. 1990; 94(14): 5502–5509. Publisher Full Text\n\nCremer D: RING - A Coordinate Transformation Program for Evaluating the Degree and Type of Puckering of a Ring Compound. Quantum Chemical Program Exchange, No. 288, 1975; 1–8. Reference Source\n\nHumphrey W, Dalke A, Schulten K: VMD: visual molecular dynamics. J Mol Graph. 1996; 14(1): 33–38, 27–8. PubMed Abstract \| Publisher Full Text\n\nCross S, Kuttel MM, Stone JE, et al.: Visualisation of cyclic and multi-branched molecules with VMD. J Mol Graph Model. 2009; 28(2): 131–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrooks BR, Brooks CL 3rd, Mackerell AD Jr, et al.: CHARMM: the biomolecular simulation program. J Comput Chem. 2009; 30(10): 1545–614. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGoecks J, Nekrutenko A, Taylor J, et al.: Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 2010; 11(8): R86. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarnett CB, Aoki-Kinoshita KF, Naidoo KJ: The Glycome Analytics Platform: an integrative framework for glycobioinformatics. Bioinformatics. 2016; 32(19): 3005–11. PubMed Abstract \| Publisher Full Text\n\nCock PJ, Antao T, Chang JT, et al.: Biopython: freely available Python tools for computational molecular biology and bioinformatics. Bioinformatics. 2009; 25(11): 1422–1423. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcNaught AD: Nomenclature of Carbohydrates (IUPAC Recommendations 1996). Pure & Appl Chem. 1996; 68(10): 1919–2008. Publisher Full Text\n\nFigueras J: Ring Perception Using Breadth-First Search. J Chem Inf Comput Sci. 1996; 36(5): 986–991. Publisher Full Text\n\nchrisbarnettster: scientificomputing/tessellate v0.3.5 (Version v0.3.5). Zenodo. 2017. Data Source\n\nEwels P, Ankenbrand MJ, Hammarén R, et al.: scientificomputing/Montage v1.4 (Version v1.4). Zenodo. 2017. Data Source" }	[ { "id": "28851", "date": "21 Dec 2017", "name": "Marcello Sega", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present two python-based packages for the analysis of ring conformations in molecular systems and for the visualization of the related data. The tessellate package implements various algorithms for the detection of cycles of different size (5 to 8 included) and represents a handy tool, which pairs nicely with its visualization counterpart, Montage, to produce and analyze aggregate data regarding the structure of the cycles. I tested the code, which is freely available, and was able to reproduce the examples reported in the article. I think that these packages will prove useful and that this article should be indexed in F1000Research.\nSome comments regarding possible (optional) improvements :\n\n1) tessellate can not only be run as a standalone command, but can also be used as a python module. This can be beneficial to the interoperability with other libraries, and probably a couple of lines could be added to the text to show an example of how to do that.\n2) if possible, it would be helpful to sort by size the elements of the histograms generated by Montage, so that at the bottom of the stack is the largest population, followed by the second-most large, and so on.\n3) regarding interoperability: output in the pandas data frame format would be probably a useful addition.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "35646", "date": "31 Jul 2018", "name": "B. Lachele Foley", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRelevant reviewer expertise: conformational properties of carbohydrates, especially puckering of 6-member rings; Linux; scientific computing, especially molecular modeling and the proper communication of simulation methods; communication of technical information to interdisciplinary and non-specialist audiences; some biochemistry of carbohydrates.\nArticle summary: The authors present a pair of software tools, Tessellate and Montage, for the analysis of ring conformations (typically referred to as ring pucker). The first tool, Tessellate, analyzes data containing coordinates of atoms bonded cyclically. The output of Tessellate can be processed by Montage to produce visual representations.\nReview summary: Overall, the article is acceptable and generally well written. The tools described are likely to be useful to persons studying ring conformations. But, a few points need to be addressed before it should be considered complete, and the article would be enhanced by some other modifications.\nAttention to these points is required:\nProvenance information should be provided for the simulation data. That the data are made available is very good, but readers also need to know where it came from. It is important that readers be able to determine the extent to which the data should be considered only as illustrations or whether they can be considered as valid scientific data. A list of reporting requirements for simulation data is recommended near the end of “Carbohydrate force fields”, available at: Foley et al1. If the data are from a separately published, peer-reviewed paper, it is sufficient to reference the paper. It is important to fully caption figures. A listing the meanings of all the pucker abbreviations used in the figures is essential. The table could be in the text or given as a key in one or more of the figures. This is for persons out of field as well as experts who are simply from another branch. For example, I do not know what UAP means; someone who has not studied the subject would be very lost. I expect that the meanings are all in referenced papers, but it is kind not to make readers consult references simply to understand such a significant element in a figure. Include addresses, and references in the text, for all websites, software and databases listed, even the ones that ‘everyone knows’. For example, although PDB is defined in the abbreviations section, there is no indication to a user how to find the Protein Data Bank. Also, VMD is referenced in a table, but it should be referenced at its first mention in the text as well. There might be other similar instances. Notes about the figures:\nThe graphs need better titles. A title like “Tessellate: Pucker series for five” can be made sense of after some thought, but one has to look around a moment to realize it means a five-membered ring, as opposed to a 5 nanosecond simulation or some such. Also, since the whole paper is about Tessellate and Montage, it is not necessary to include the word Tessellate unless it is being compared to something else. A title like “Pucker results for a five-membered ring” would be better. Please explicitly say somewhere that you are assuming a constant puckering amplitude. A five-membered ring requires two parameters, a six-membered ring requires three, and so forth.\nRelated to that, the phrase “Numeric Pucker 1D” in Figure 2 is not defined.\n\nThe x-axis for the top graph in Figure 2 says “Time or sequence index.” Which is it? If it is a time, what is the unit? Increase the font size on the axes where they are very small. I would make them at least the size of the title of the first graph in Figure 2.\n\nAttention to these points would enhance the paper:\nA little reorganization in the Methods section would make the article more accessible and possibly open the tools up for a wider audience. In particular, the subsection titled Implementation is confusing. It starts with a prerequisite (use Linux), which probably goes in the Operation section. Then, it lists a number of features. While the analysis method used for each feature is also described, which is very good, I think the section would be better titled “Available Analytical Protocols” or even “Features”, and the line about Linux removed as it is made clear in Operation. In cases like those presented in Figure 4, I would find it more useful to present the data as a fraction or percentage rather than raw counts. But, this is merely my opinion and recommendation. It is certainly unreasonable to expect every paper to reference every other paper with any relevance, but you might take a look at one that I have considerable knowledge of, “BFMP: A Method for Discretizing and Visualizing Pyranose Conformations”, available here: Makeneni et al2. For that paper, we spent a lot of time thinking of ways to communicate information about pucker in 6-membered rings, and I think we came up with some nice graphical methods. You might consider including some of them regardless whether you like the classification scheme.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2113
https://f1000research.com/articles/6-1515/v1	18 Aug 17	{ "type": "Research Article", "title": "RNAi targeting Caenorhabditis elegans α-arrestins marginally affects lifespan", "authors": [ "Sangsoon Park", "Yoonji Jung", "Seon Woo A. An", "Heehwa G. Son", "Wooseon Hwang", "Dongyeop Lee", "Murat Artan", "Hae-Eun H. Park", "Dae-Eun Jeong", "Yujin Lee", "Seung-Jae V. Lee", "Sangsoon Park", "Yoonji Jung", "Seon Woo A. An", "Heehwa G. Son", "Wooseon Hwang", "Dongyeop Lee", "Murat Artan", "Hae-Eun H. Park", "Dae-Eun Jeong", "Yujin Lee" ], "abstract": "Background: α-arrestins are a family of proteins that are implicated in multiple biological processes, including metabolism and receptor desensitization. Methods: Here, we sought to examine the roles of α-arrestins in the longevity of Caenorhabditis elegans through an RNA interference screen. Results: We found that knocking down each of 24 out of total 29 C. elegans α-arrestins had small or no effects on lifespan. Thus, individual C. elegans α-arrestins may have minor effects on longevity. Conclusions: This study will provide useful information for future research on the functional role of α-arrestins in aging and longevity.", "keywords": [ "C. elegans", "α-arrestin", "insulin/IGF-1 signaling", "aging", "lifespan" ], "content": "Introduction\n\nα-arrestins are a family of proteins that contain arrestin domains whose sequences and structures have similarities with those of classical visual and β-arrestins1–3. α-arrestins are considered as ancestral forms of arrestins because their orthologs exist in fungi, including yeast, which do not have visual or β-arrestins1,4. Several mammalian α-arrestins modulate metabolism and receptor desensitization5,6, but much remains to be elucidated concerning the functions of α-arrestins in many organisms. The human genome encodes 6 α-arrestins and 4 visual or β-arrestins. Interestingly, the simple roundworm Caenorhabditis elegans genome contains 29 α-arrestin and 1 β-arrestin genes1,7. Therefore, the C. elegans system provides opportunities for the genetic analysis of α-arrestins in various aspects of physiology both individually and combinatorially. However, information regarding the functions of C. elegans α-arrestins is limited8,9.\n\nC. elegans is an excellent genetic model organism that has been exploited for studying conserved biological processes, including apoptosis, behavior, development and aging. In particular, its short lifespan in combination with genetic amenability has made C. elegans one of the most popular models for research on aging and longevity10,11. Many factors, including components in insulin/insulin-like growth factor 1 (IGF-1) signaling (IIS), have been identified as lifespan and aging regulators in C. elegans11–13. For example, genetic inhibition of IIS components, such as DAF-2/insulin/IGF-1 receptor, robustly extends lifespan and delays physiological aging through up-regulating transcription factors, including DAF-16/FOXO11–13. Importantly, the roles of these aging-regulatory factors in C. elegans have been shown to be conserved in other species, including Drosophila and mammals12,13. One of the powerful ways to identify novel factors that influence aging is by employing genetic screens, such as an RNA interference (RNAi) screen. We previously identified several genetic factors, including RNA helicases, that modulate longevity in C. elegans, through targeted or genome-wide RNAi screens14–17. Because of its robust longevity phenotype that confers sensitivity to changes in lifespan, daf-2/insulin/IGF-1 receptor mutants serve as an excellent platform for the identification of novel lifespan-regulating factors17,18.\n\nIn this study, we aimed to determine whether any α-arrestins played a role in the lifespan regulation of wild-type or daf-2 mutants. We performed a lifespan assay-based RNAi screen targeting 24 out of 29 C. elegans α-arrestins. We found that knocking down each of the α-arrestins had small or no effects on the lifespan of wild-type and daf-2 mutants. Thus, C. elegans α-arrestins may play minor or modulatory roles in lifespan regulation. Based on our results, it will be important to test the roles of α-arrestins in combinatorial manners and/or by using strong loss-of-function mutations in future research.\n\n\nMethods\n\nAll strains were maintained as previously described19. The following strains were used in this study: N2 wild-type, CF1041 daf-2(e1370) III outcrossed six times to N2.\n\nThe protein sequences of 27 α-arrestins, except arrd (arrestin domain protein)-20 and arrd-21, were obtained from Wormbase (www.wormbase.org, version WS259). The protein sequences of arrd-20 and arrd-21 were obtained from Ensembl (http://www.ensembl.org, release 89). The phylogenetic tree of 29 α-arrestins in C. elegans was generated using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/)20 and re-visualized using the Dendroscope 3 (version 3.5.9)21. For the α-arrestins that have multiple isoforms, isoform a was used for the analysis.\n\nTwenty one RNAi clones that target C. elegans α-arrestin genes were used from two commercial C. elegans RNAi feeding libraries. Specifically, RNAi clones targeting arrd-2, arrd-6, arrd-7, arrd-8, arrd-9, arrd-10, arrd-13, arrd-16, arrd-18, arrd-23, arrd-24, arrd-25, arrd-28 and ttm-2 (toxin-regulated targets of MAPK 2) were obtained from Ahringer laboratory library (Geneservice Ltd., Cambridge, UK), while those against arrd-1, arrd-3, arrd-4, arrd-5, arrd-14, arrd-15 and arrd-19 were from Vidal laboratory library (Source BioScience, Nottingham, UK). Three RNAi clones targeting arrd-11, arrd-17, and arrd-26 were generated by molecular cloning using infusion recombinase (EZ-FusionTM Cloning Kit, Enzynomics, Daejeon, South Korea). The N2 genomic DNA was obtained through the lysis of worms using proteinase K (Invitrogen, Carlsbad, CA, USA) and N2 complementary DNA was obtained from RNA extraction using RNAiso Plus (Takara, Shiga, Japan) followed by reverse transcription using ImProm-II reverse transcriptase (Promega, Madison, WI, USA, USA). The infusion reaction between PCR products and pL4440 plasmids (Fire lab C. elegans vector kit, 1999) digested with HindIII (New England Biolabs, Ipswich, MA, USA) and Acc65I (New England Biolabs) was followed by transformation of in-house competent E. coli HT115 cells and by selection of positive clones on ampicillin (USB, Santa Clara, CA, USA)-containing LB plates. Primers (CosmoGenetech, Seoul, South Korea) that were used to amplify coding regions of arrd-11 from N2 genomic DNA, and those of arrd-17 and arrd-26 from N2 complementary DNA are as follows: forward primer 5’-GAATTCGATATCAAGCTCCCTCGTGCAAATTAGGAAA-3’ and reverse primer 5’-CTATAGGGCGAATTGGGGTTCCTCCCACTCCATACA-3’ for arrd-11; forward primer 5’-GAATTCGATATCAAGCTATGGTGCAGTTAGATCGTTTTG-3’ and reverse primer 5’-CTATAGGGCGAATTGGTTAATCGGTATAAAATGG-3’ for arrd-17; forward primer 5’-GAATTCGATATCAAGCTATGAAGGTCGATTACTTCG-3’ and reverse primer 5’- CTATAGGGCGAATTGGCTACTTCTCGGAGCCATTTG-3’ for arrd-26. The sequences of all these 24 α-arrestin RNAi clones were confirmed using DNA sequencing (Solgent, Daejeon, South Korea) before lifespan assays. RNAi clones targeting remaining five α-arrestin genes were not generated.\n\nThe RNAi screen employing lifespan assay was performed as previously described17. Briefly, E. coli HT115 bacteria that expressed specific double-stranded RNAs (dsRNAs) were cultured overnight at 37°C in LB media containing 50 μg/ml ampicillin (USB). The cultured bacteria were seeded onto nematode growth media plates containing 50 μg/ml ampicillin and incubated overnight at 37°C. The seeded bacteria were treated with 1 mM isopropyl β-D-1-thiogalactopyranoside (Gold Biotechnology, St. Louis, MO, USA) and incubated at room temperature for approximately 24 h to induce dsRNAs. Age-synchronized wild-type N2 and daf-2(e1370) mutant animals were grown on RNAi plates from embryo to L4 stage at 20°C. Worms were then transferred onto RNAi plates containing 5 μM 5-fluoro-2’deoxyuridine (FUdR; Sigma, St. Louis, MO, USA), which prevents eggs from hatching, at young (day 1) adult stage, and transferred again to new FUdR-containing RNAi plates after 1 or 2 days. All lifespan assays were performed at 20°C as duplicates by two independent researchers. The survival of worms was determined by gently touching worms with a platinum wire. Worms that did not respond were counted as dead worms and removed from the plates. Worms that crawled off the plates, burrowed into the agar media, or displayed internal hatching or vulval rupture were censored, but included in the statistical analysis. Lifespan data from two independent lifespan experiments within the experimental sets were pooled and used for statistical analysis (see Dataset 1–Dataset 3). OASIS 2 (Online Application for Survival Analysis 2; https://sbi.postech.ac.kr/oasis2)22 was used for the statistical analysis of lifespan results. P values were calculated using long-rank (Mantel-Cox method) test.\n\n\nResults\n\nWe measured the lifespan of wild-type and long-lived daf-2 mutant animals upon knocking down each of 24 of 29 genes encoding putative α-arrestin proteins (Figure 1A). We used daf-16 RNAi that largely suppressed the longevity of daf-2(-) mutants as a positive control (Figure 1B, Figure S1A, Table S1)17,18. We found that knockdown of each of individual α-arrestin genes tended to slightly reduce lifespan in wild-type or in daf-2(-) mutants (Figure 1B, Figure S1B–U, Table S1). Out of the 24 RNAi clones, RNAi targeting arrd (arrestin domain protein)-13, arrd-16, arrd-23, arrd-24 or arrd-25 in wild-type, and RNAi against arrd-1, arrd-2, arrd-5, arrd-24 or arrd-28 in daf-2(-) mutant animals decreased lifespan by more than 5% (Figure 1B–E, Figures S1B, D, K, Q, R, T, Table S1). Specifically, arrd-16 RNAi decreased lifespan in wild-type by 9%, and arrd-1 RNAi decreased lifespan in daf-2(-) mutants by 7% (Figure 1B and C). In addition, RNAi targeting arrd-24 decreased the lifespan of wild-type and daf-2 mutant animals by 11% and 6%, respectively (Figure 1E). In contrast, arrd-3 RNAi increased the lifespan of wild-type animals by 10% (Figure 1F). Overall, genetic inhibition of individual α-arrestin genes using RNAi appears to have minor effects on C. elegans lifespan.\n\n(A) A phylogenetic tree showing C. elegans α-arrestin family members. Asterisks (*) indicate α-arrestins that were not examined in this study and number signs (#) indicate predicted pseudogenes. (B) Each circle represents percent mean lifespan change by treatment with individual α-arrestin gene RNAi in wild-type (WT) and daf-2(e1370) (daf-2(-)) mutants. Red triangles indicate lifespan changes by daf-16 RNAi, which was used as a positive control. (C–F) Lifespan curves of WT and daf-2(-) animals treated with RNAi targeting arrd-16 (C), arrd-1 (D), arrd- 24 (E), and arrd-3 (F). See Table S1 for statistical analysis.\n\n\nDiscussion\n\nThe lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. As knocking down each of several α-arrestins resulted in minor changes in lifespan, these C. elegans α-arrestins may play modulatory roles in longevity regulation. RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes for various reasons. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons7,9,23–25, which are refractory to RNAi26–28. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals28–30, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. Another possibility is that C. elegans α-arrestins may have functional redundancy, considering the large number of the α-arrestin family members in C. elegans and their sequence similarity1,7. In addition, some α-arrestins may mostly function by modulating the action of their interacting proteins6. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.\n\nIn mammals, several α-arrestins are implicated in metabolic regulation5. TXNIP (thioredoxin-interacting protein), an inhibitor of thioredoxin in mammals31–33, is a crucial negative regulator of glucose uptake34,35. ARRDC4 inhibits glucose uptake in cultured mammalian cells as well35, and ARRDC3 deficiency protects against obesity in male mice through increasing energy expenditure36. Because metabolism is closely associated with aging37, it will be interesting to test whether α-arrestins in complex metazoans play roles in aging via regulating metabolism.\n\n\nData availability\n\nDataset 1. Kaplan-Meier estimator of RNAi lifespan experiments. Kaplan-Meier estimate values were calculated from pooled lifespan data of two independent lifespan experiments using OASIS2 (https://sbi.postech.ac.kr/oasis2/). ‘At risk’ indicates the number of individuals at risk just prior to a specific time point. ‘S_hat’ indicates Kaplan-Meier estimate of survival function, ‘Var S_hat’ indicates variance of ‘S_hat’, and ‘SE(S)’ indicates standard error of ‘S_hat’. doi, 10.5256/f1000research.12337.d17315838\n\nDataset 2. Mean lifespan and mortality rates of RNAi experiment results. Mean lifespan, ages at different percent mortality, linear interpolation of mortality curve at specific mortality rate were calculated from pooled lifespan data of two independent lifespan experiments. Data were obtained using OASIS2 (https://sbi.postech.ac.kr/oasis2/). doi, 10.5256/f1000research.12337.d17315939\n\nDataset 3. Statistical analysis of lifespan data. Statistical test results (Chi square, p value, Bonferroni p value) were calculated between ‘condition 1’ and ‘condition 2’. Test results were obtained using OASIS2 (https://sbi.postech.ac.kr/oasis2/). doi, 10.5256/f1000research.12337.d17316040", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the Korean Government (MSIP) through the National Research Foundation of Korea (NRF) [NRF-2016R1E1A1A01941152] funded to S-J.V.L.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank the Lee laboratory members and Dr. Young Kwon for critical comments on the manuscript.\n\n\nSupplementary material\n\nFigure S1. RNAi knockdown of each Caenorhabditis elegans α-arrestin had minor effects on the lifespan of wild-type or daf-2(-) mutants. (A–U) Lifespan curves of wild-type (WT) and daf-2(e1370) (daf-2(-)) mutants treated with RNAi targeting daf-16 (A), which was used as a positive control, arrd-2 (B), arrd-4 (C), arrd-5 (D), arrd-6 (E), arrd-7 (F), arrd-8 (G), arrd-9 (H), arrd-10 (I), arrd-11 (J), arrd-13 (K), arrd-14 (L), arrd-15 (M), arrd-17 (N), arrd-18 (O), arrd-19 (P), arrd-23 (Q), arrd-25 (R), arrd-26 (S), arrd-28 (T) and ttm-2 (U). See Table S1 for statistical analysis and for additional repeats of lifespan data using daf-16 RNAi.\n\nClick here to access the data.\n\nTable S1. Lifespan data of Caenorhabditis elegans α-arrestin RNAi clones. Lifespan data within the solid lines indicate the pooled experimental datasets that are performed at the same time by two independent researchers. P values were calculated within the sets using log-rank (Mantel-Cox) method22. Percent (%) changes and p values for the lifespan data of specific α-arrestin RNAi-treated wild-type (WT) and daf-2(e1370) worms were calculated against those of control RNAi-treated WT and daf-2(e1370) worms within the sets, respectively. Percent (%) changes and p values for the lifespan data of control RNAi-treated daf-2(e1370) mutants were calculated against those of control RNAi-treated WT worms. SEM represents standard error of the mean, and 75th percentile indicates the age (days) at 75% mortality.\n\nClick here to access the data.\n\n\nReferences\n\nAlvarez CE: On the origins of arrestin and rhodopsin. BMC Evol Biol. 2008; 8: 222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAubry L, Guetta D, Klein G: The arrestin fold: variations on a theme. Curr Genomics. 2009; 10(2): 133–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPolekhina G, Ascher DB, Kok SF, et al.: Structure of the N-terminal domain of human thioredoxin-interacting protein. Acta Crystallogr D Biol Crystallogr. 2013; 69(Pt 3): 333–344. PubMed Abstract \| Publisher Full Text\n\nBecuwe, M, Herrador A, Haguenauer-Tsapis R, et al.: Ubiquitin-mediated regulation of endocytosis by proteins of the arrestin family. Biochem Res Int. 2012; 2012: 242764. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatwari P, Lee RT: An expanded family of arrestins regulate metabolism. Trends Endocrinol Metab. 2012; 23(5): 216–222. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPuca L, Brou C: Α-arrestins - new players in Notch and GPCR signaling pathways in mammals. J Cell Sci. 2014; 127(Pt 7): 1359–1367. PubMed Abstract \| Publisher Full Text\n\nHobert O: The neuronal genome of Caenorhabditis elegans. WormBook. 2013: 1–106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuffman DL, Abrami L, Sasik R, et al.: Mitogen-activated protein kinase pathways defend against bacterial pore-forming toxins. Proc Natl Acad Sci USA. 2004; 101(30): 10995–11000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJee C, Choi TW, Kalichamy K, et al.: CNP-1 (ARRD-17), a novel substrate of calcineurin, is critical for modulation of egg-laying and locomotion in response to food and lysine sensation in Caenorhabditis elegans. J Mol Biol. 2012; 417(3): 165–178. PubMed Abstract \| Publisher Full Text\n\nTissenbaum HA: Using C. elegans for aging research. Invertebr Reprod Dev. 2015; 59(sup1): 59–63. Publisher Full Text \| Free Full Text\n\nAn SWA, Artan M, Park S, et al.: Longevity Regulation by Insulin/IGF-1 Signalling. In Ageing: Lessons from C. elegans. A. Olsen and M.S. Gill, Editors. Springer International Publishing. 2017; 63–81. Reference Source\n\nAltintas O, Park S, Lee SJ: The role of insulin/IGF-1 signaling in the longevity of model invertebrates, C. elegans and D. melanogaster. BMB Rep. 2016; 49(2): 81–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKenyon CJ: The genetics of ageing. Nature. 2010; 464(7288): 504–512. PubMed Abstract \| Publisher Full Text\n\nLee SJ, Hwang AB, Kenyon C: Inhibition of respiration extends C. elegans life span via reactive oxygen species that increase HIF-1 activity. Curr Biol. 2010; 20(23): 2131–2136. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHwang W, Artan M, Seo M, et al.: Inhibition of elongin C promotes longevity and protein homeostasis via HIF-1 in C. elegans. Aging Cell. 2015; 14(6): 995–1002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLee D, Jeong DE, Son HG, et al.: SREBP and MDT-15 protect C. elegans from glucose-induced accelerated aging by preventing accumulation of saturated fat. Genes Dev. 2015; 29(23): 2490–2503. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeo M, Seo K, Hwang W, et al.: RNA helicase HEL-1 promotes longevity by specifically activating DAF-16/FOXO transcription factor signaling in Caenorhabditis elegans. Proc Natl Acad Sci U S A. 2015; 112(31): E4246–E4255. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSamuelson AV, Carr CE, Ruvkun G: Gene activities that mediate increased life span of C. elegans insulin-like signaling mutants. Genes Dev. 2007; 21(22): 2976–2994. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStiernagle T: Maintenance of C. elegans. WormBook. 2006: 1–11. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSievers F, Higgins DG: Clustal omega. Curr Protoc Bioinformatics. 2014; 48: 3.13.1–3.13.16. PubMed Abstract \| Publisher Full Text\n\nHuson DH, Scornavacca C: Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol. 2012; 61(6): 1061–1067. PubMed Abstract \| Publisher Full Text\n\nHan, SK, Lee D, Lee H, et al.: OASIS 2: online application for survival analysis 2 with features for the analysis of maximal lifespan and healthspan in aging research. Oncotarget. 2016; 7(35): 56147–56152. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKunitomo H, Uesugi H, Kohara Y, et al.: Identification of ciliated sensory neuron-expressed genes in Caenorhabditis elegans using targeted pull-down of poly(A) tails. Genome Biol. 2005; 6(2): R17. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLynch AS, Briggs D, Hope IA: Developmental expression pattern screen for genes predicted in the C. elegans genome sequencing project. Nat Genet. 1995; 11(3): 309–313. PubMed Abstract \| Publisher Full Text\n\nMounsey A, Bauer P, Hope IA: Evidence suggesting that a fifth of annotated Caenorhabditis elegans genes may be pseudogenes. Genome Res. 2002; 12(5): 770–775. PubMed Abstract \| Free Full Text\n\nTimmons L, Court DL, Fire A: Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene. 2001; 263(1–2): 103–112. PubMed Abstract \| Publisher Full Text\n\nKamath RS, Martinez-Campos M, Zipperlen P, et al.: Effectiveness of specific RNA-mediated interference through ingested double-stranded RNA in Caenorhabditis elegans. Genome Biol. 2001; 2(1): RESEARCH0002. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKennedy S, Wang D, Ruvkun G: A conserved siRNA-degrading RNase negatively regulates RNA interference in C. elegans. Nature. 2004; 427(6975): 645–649. PubMed Abstract \| Publisher Full Text\n\nSimmer F, Tijsterman M, Parrish S, et al.: Loss of the putative RNA-directed RNA polymerase RRF-3 makes C. elegans hypersensitive to RNAi. Curr Biol. 2002; 12(15): 1317–1319. PubMed Abstract \| Publisher Full Text\n\nSijen T, Fleenor J, Simmer F, et al.: On the role of RNA amplification in dsRNA-triggered gene silencing. Cell. 2001; 107(4): 465–476. PubMed Abstract \| Publisher Full Text\n\nJunn E, Han SH, Im JY, et al.: Vitamin D3 up-regulated protein 1 mediates oxidative stress via suppressing the thioredoxin function. J Immunol. 2000; 164(12): 6287–6295. PubMed Abstract \| Publisher Full Text\n\nNishiyama A, Matsui M, Iwata S, et al.: Identification of thioredoxin-binding protein-2/vitamin D3 up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem. 1999; 274(31): 21645–21650. PubMed Abstract \| Publisher Full Text\n\nYamanaka H, Maehira F, Oshiro M, et al.: A possible interaction of thioredoxin with VDUP1 in HeLa cells detected in a yeast two-hybrid system. Biochem Biophys Res Commun. 2000; 271(3): 796–800. PubMed Abstract \| Publisher Full Text\n\nParikh H, Carlsson E, Chutkow WA, et al.: TXNIP regulates peripheral glucose metabolism in humans. PLoS Med. 2007; 4(5): e158. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatwari P, Chutkow WA, Cummings K, et al.: Thioredoxin-independent regulation of metabolism by the alpha-arrestin proteins. J Biol Chem. 2009; 284(37): 24996–25003. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPatwari P, Emilsson V, Schadt EE, et al.: The arrestin domain-containing 3 protein regulates body mass and energy expenditure. Cell Metab. 2011; 14(5): 671–683. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFinkel T: The metabolic regulation of aging. Nat Med. 2015; 21(12): 1416–1423. PubMed Abstract \| Publisher Full Text\n\nPark S, Jung Y, An SWA, et al.: Dataset 1 in: RNAi targeting Caenorhabditis elegans. α-arrestins marginally affects lifespan. F1000Research. 2017. Data Source\n\nPark S, Jung Y, An SWA, et al.: Dataset 2 in: RNAi targeting Caenorhabditis elegans. α-arrestins marginally affects lifespan. F1000Research. 2017. Data Source\n\nPark S, Jung Y, An SWA, et al.: Dataset 3 in: RNAi targeting Caenorhabditis elegans. α-arrestins marginally affects lifespan. F1000Research. 2017. Data Source" }	[ { "id": "25290", "date": "04 Sep 2017", "name": "Alessandro Bitto", "expertise": [ "Reviewer Expertise Biology of Aging" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research.\n\nMajor concern: The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses.\n\nMinor concerns:\n\n1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans.\n\n2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion1. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results.\n\n3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously.\n\n4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [ { "c_id": "3112", "date": "14 Nov 2017", "name": "Seung-Jae V Lee", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Dr. Alessandro Bitto and Dr. Jason Pitt, We greatly appreciate the reviewers’ valuable comments on our manuscript. We agree with all the reviewers’ comments and modified our manuscript to describe the limitations of our studies raised by the reviewers. We would like to mention that the main purpose of publishing our current manuscript is to provide our data to other researchers who are interested in the characterization of C. elegans arrd genes, instead of keeping our current data unpublished. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research. Reviewers’ comments: In the manuscript RNAi targeting Caenorhabditis elegans alpha-arrestins marginally affects lifespan Park and co-workers provide largely negative results from lifespan experiments following RNAi knockdown of most of the worm alpha-arrestins. While we welcome the effort to publish negative results, there is one major concern with the experimental approach and several minor concerns with the interpretation of the results that should be addressed before the manuscript is fully accepted for indexing in F1000Research. Major concern: The authors data is almost entirely negative: there were only 4 clones which had a statistically significant effect. The authors attempt to address this concern by providing RNAi data for daf-16, which is known to strongly suppress the lifespan extension of daf-2(e1370) as well as decreasing N2 lifespan. However, the authors must demonstrate that alpha-arrestin RNAi knockdown is functioning. As suggested by the authors, there is some evidence that alpha-arrestins are expressed in neurons which are notoriously refractory to RNAi. The authors must show that they can use RNAi to knockdown alpha-arrestins by QRTPCR analysis. The authors could also address this concern by performing the RNAi in one of the mutant strains in which RNAi works in neurons and report if the lifespan still does not change, as they suggest in the discussion. We believe that providing these data is necessary to correctly interpret the results from the lifespan analyses. > We completely agree with the reviewers for the comment. It would have been great if we had performed qRTPCR for validating the RNAi efficiency, while we were doing lifespan assays. As our experiments were done as an RNAi screen, our initial plan was validating RNAi efficiency and using RNAi-sensitive mutants if we had identified some positive RNAi clones. However, as the reviewers pointed out, our screen results were negative. We currently do not have any plan for following up this project and just want to inform other researchers of our screen data. Therefore, instead of putting efforts on this project retrospectively, we emphasized the limitations of our data raised by the reviewers to inform the readers of pitfalls by revising the manuscript as follows, In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons7, 9, 24-26, which are refractory to RNAi 27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals 29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.” Minor concerns: 1. In the text the authors speak broadly of individual arrestins having no role in longevity however the authors only tested 24 of the 29 arrestins in the worm. If the authors had limited themselves to only those clones available in commercial RNAi libraries this would be understandable, however the authors did construct 3 new RNAi feeding constructs. This brings up the question to why the authors did not prepare the remaining 5 constructs. If the authors have reason to exclude those genes from their analysis (ie. they are pseudogenes, not expressed, etc) they should discuss these reasons in the text. Besides, there is the possibility that one of the missing constructs may indeed have major effects on lifespan in C. elegans. > We appreciate the reviewers’ comment. We initially aimed at obtaining all the α arrestin RNAi clones that are not commercially available except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones for arrd-12, arrd-22 and arrd-27 because of unknown technical problems. We agree with the reviewers’ point that these three genes that were not tested may have major effects on lifespan. We addressed this issue in Methods for RNAi clones as well as in Discussion as follows, In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.” In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.” 2. The title of the manuscript implies there are more than one result than was observed. The simpler conclusion from the results provided is that individual alpha-arrestin knockdown has no effect of lifespan. This is the fundamental observation of the paper considering that most of them (20 out of 24) had no effect and that 4 of them had such an extremely small effect that it is arguable that the sample size is insufficient to support any conclusion1. We understand the authors' motivation to make the manuscript sound more appealing, however, their interpretation is disingenuous, given their results. > We thank the reviewers’ comment on the title and changed the title as follows, Title: “RNAi targeting Caenorhabditis elegans α-arrestins has small or no effects on lifespan” 3. Because alpha-arrestins share significant sequence homology the authors should make some mention as to the likelihood that RNAi of any one arrestin would have off target effects on other arrestins. The authors could easily address this question experimentally with QRTPCR analysis. This is particularly relevant as one of the main conclusions of the manuscript is that future alpha-arrestin studies should knockdown multiple alpha-arrestins simultaneously. > We agree with the reviewers that the RNAi targeting each of α-arrestin genes may have caused off-target effects on other α-arrestin genes because of sequence similarities. We performed off-target prediction using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap), and at least with the prediction, the RNAi clones used in our screen do not appear to have major off-target effects on any other genes including α-arrestin genes, except arrd-19 RNAi which may also target arrd-21 which is a predicted pseudogene. Although qRT-PCR will be a straightforward way as the reviewers suggested, again currently we do not have any plan on following up this project and we therefore instead acknowledged the limitation. We added the prediction results in the Supplementary Table S1 and described this issue in the Methods, RNAi clones part as follows, In Methods, RNAi clones: “All RNAi clones used in our screen were examined for their potential off-target effects by using Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)22, and no significant off-target was predicted except arrd-19 RNAi, which may additionally target a predicted pseudogene arrd-21 (Table S1). Experimental validation by qRT-PCR will be necessary to completely exclude the possible off-target effects by RNAi clones.” In Supplementary material: “Table S1. Possible RNAi target prediction. RNAi clones used in the screen were examined for their possible off-targets by Clone Mapper (http://bioinformatics.lif.univ-mrs.fr/RNAiMap)22. For RNAi clone names, ‘mv’ indicates a RNAi clone from Vidal library and ‘sjj’ indicates a RNAi clone from Ahringer library. RNAi clones generated by us for this study are labeled as ‘Lee lab’. Scores for potential target transcripts were calculated as described previously22 and were rounded off to the second digit after the decimal point.” In Reference: 22. Thakur, N., et al., Clone Mapper: An Online Suite of Tools for RNAi Experiments in Caenorhabditis elegans. G3 (Bethesda), 2014. 4(11): p. 2137-2145.23. 4. Given that the authors observed the RNAi treated animals for weeks or months (in daf-2) it is surprising that no other behavioral or physical phenotypes from the alpha-arrestin RNAi were observed during the course of the analysis. If the authors truly did not observe any other phenotypes they should state it briefly in the text. > We appreciate the reviewers’ comment. As our main interests were lifespan changes from a large scale screen, we did not pay attention to other phenotypes. Nevertheless, one noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantitate the results. We described this issue in Methods as follows, In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”" } ] }, { "id": "25722", "date": "06 Sep 2017", "name": "Roger Pocock", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan.\n\nConcerns: One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan.\n\nIn parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously.\n\nThe authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined.\n\nThe authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3113", "date": "14 Nov 2017", "name": "Seung-Jae V Lee", "role": "Author Response F1000Research Advisory Board Member", "response": "Dear Dr. Roger Pocock, We sincerely appreciate your valuable comments on our manuscript. We agree with all your points and revised our manuscript to describe the limitations of this study. We would like to mention that the main motivation for publishing this manuscript is to provide our negative results to researchers who are interested in characterizing α-arrestins in C. elegans. Our point-by-point responses are described below. We hope you find our revised manuscript is suitable for publication in F1000Research. In this manuscript Park et al. performed RNAi knockdown of 24 of the 29 alpha-arrestin genes in C. elegans to ask whether they are important for lifespan regulation in wild type and daf-2 mutant animals. Their data shows that, in their experimental conditions, most alpha-arrestin genes are individually not important for regulating lifespan. Concerns: One major issue with the study is whether RNAi knockdown of the alpha-arrestin genes is working. As some alpha-arrestins are expressed in the nervous system, which is refractory to RNAi, this is a concern. To determine whether the RNAi knockdown is working then Q-RTPCR should be performed. However, this is not a small task for 24 genes, therefore, further experiments using sensitized genetic backgrounds for RNAi or elevated temperatures may tease out phenotypes for some alpha-arrestin genes. This could particularly be performed for the alpha-arrestin genes that when knocked down engender a slight effect on lifespan. > We agree with the reviewer’s comment that the RNAi knockdown efficiency is a major concern in interpreting our screen results. It would have been great if we had performed qRT-PCR analysis. However, as the reviewers pointed out, this is not a small task. We currently do not have any plan for following up this project and want to inform other researchers of our screen data. Therefore, we further emphasized the pitfall of our results in the Discussion as follows, In Discussion: “The lifespan-regulatory functions of α-arrestins remain largely unexplored at the organism level. Here we showed that RNAi knockdown of individual C. elegans α-arrestins had small or no effects on lifespan in wild-type or daf-2 mutants. Our study has limitations that need to be considered for interpretation. First, RNAi targeting some α-arrestins might be insufficient for causing strong lifespan phenotypes. This may be because many of C. elegans α-arrestins are predicted to be expressed in neurons7, 9, 24-26, which are refractory to RNAi27-29. Lifespan assays using RNAi-hypersensitive mutants, including rrf-3(-) and eri-1(-) animals29-31, treated with α-arrestin RNAi, or using α-arrestin null mutants will help address this issue. In addition, as we did not test whether RNAi targeting each of α-arrestin genes was effective by using quantitative RT-PCR, our negative data should be interpreted with caution.” In parallel to this, the authors could align the alpha-arrestin gene sequences to ask if there is a common sequence that could be used by RNAi targeting to knockdown multiple alpha-arrestin genes simultaneously. > We appreciate the reviewer’s comment. The α-arrestin family members share arrestin-domains (O. Hobert, Wormbook, 2013). Thus, it will be intriguing if we could examine whether targeting arrestin domain for RNAi to knock down multiple arrd genes simultaneously and if multiple arrd gene knock down has effects on lifespan. We modified our manuscript to address this issue in Discussion as follows, In Discussion: “Second, C. elegans α-arrestins may have functional redundancy considering the large number of the α-arrestin family members in C. elegans and their sequence similarity1, 7, which may obscure examining the functional importance of each α-arrestin. In addition, some α-arrestins may mostly function by modulating their interacting proteins6. In this case, genetic inhibition of α-arrestins may rather subtly affect the functions of their interacting partners that directly regulate physiology, such as aging and longevity, causing weak or no phenotypes. Thus, it will be interesting to test the effects of simultaneous inhibition of α-arrestins possibly through targeting the arrestin domain, and to identify and to functionally characterize proteins that bind C. elegans α-arrestins.” The authors knockdown of 24 of the 29 alpha-arrestin genes. I wonder why they didn’t generate RNAi clones for the remaining 5, especially since they did generate 3 other constructs for their analysis. Potentially, the important alpha-arrestin genes is amongst those not examined. > We appreciate the reviewers’ comment. We aimed at obtaining all the α arrestin RNAi clones that are not commercially available, except two predicted pseudogenes, arrd-20 and arrd-21 (Wormbase, version WS259). We successfully constructed three RNAi clones targeting arrd-11, arrd-17 and arrd-26, but could not obtain the other three RNAi clones, targeting arrd-12, arrd-22 and arrd-27. We addressed this issue in Methods for RNAi clones and in Discussion as follows, In Methods, RNAi clones: “RNAi clones for arrd-12, arrd-22, and arrd-27 were not generated because of unknown technical problems and were not included in the lifespan screen. arrd-20 and arrd-21 are predicted to be pseudogenes (Wormbase, version WS259) and were excluded from our screen.” In Discussion: “Third, it is possible that three α-arrestins, arrd-12, arrd-22 and arrd-27, which were not tested in our screen, may play crucial roles in lifespan regulation. Thus, it will be important to examine if genetic inhibition of each of these three α-arrestin genes affects lifespan in future studies.” The authors did not report any other behavioural or morphological phenotypes observed by alpha-arrestin gene knockdown. Any information on observed phenotypes would be of interest to the research community. > We thank the reviewer for the valuable comment. One noticeable phenotype we observed was that the eggs laid by worms fed with ttm-2 RNAi bacteria hatched more frequently than control eggs on FUdR-treated plates, although we did not quantify the results. We described this issue in Methods as follows, In Methods, RNAi screen using lifespan assays: “Eggs laid by ttm-2 RNAi-treated worms hatched more frequently than control eggs on FUdR-containing plates in two independent lifespan experiments.”" } ] } ]	1	https://f1000research.com/articles/6-1515
https://f1000research.com/articles/6-1601/v1	30 Aug 17	{ "type": "Research Article", "title": "In silico analysis of natural compounds targeting structural and nonstructural proteins of chikungunya virus", "authors": [ "Jaspreet Jain", "Anchala Kumari", "Pallavi Somvanshi", "Abhinav Grover", "Somnath Pai", "Sujatha Sunil", "Jaspreet Jain", "Anchala Kumari", "Pallavi Somvanshi", "Abhinav Grover", "Somnath Pai" ], "abstract": "Background: Chikungunya fever presents as a high-grade fever during its acute febrile phase and can be prolonged for months as chronic arthritis in affected individuals. Currently, there are no effective drugs or vaccines against this virus. The present study was undertaken to evaluate protein-ligand interactions of all chikungunya virus (CHIKV) proteins with natural compounds from a MolBase library in order to identify potential inhibitors of CHIKV. Methods: Virtual screening of the natural compound library against four non-structural and five structural proteins of CHIKV was performed. Homology models of the viral proteins with unknown structures were created and energy minimized by molecular dynamic simulations. Molecular docking was performed to identify the potential inhibitors for CHIKV. The absorption, distribution, metabolism and excretion (ADME) toxicity parameters for the potential inhibitors were predicted for further prioritization of the compounds. Results: Our analysis predicted three compounds, Catechin-5-O-gallate, Rosmarinic acid and Arjungenin, to interact with CHIKV proteins; two (Catechin-5-O-gallate and Rosmarinic acid) with capsid protein, and one (Arjungenin) with the E3. Conclusion: The compounds identified show promise as potential antivirals, but further in vitro studies are required to test their efficacy against CHIKV.", "keywords": [ "In silico analysis", "Chikungunya virus", "natural compounds", "CHIKV E3 protein", "CHIKV Capsid protein", "Docking", "ADME", "Ligand-Protein Interaction" ], "content": "Introduction\n\nChikungunya virus (CHIKV) is an alphavirus belonging to the Togaviridae family1. These are small, spherical, enveloped viruses that constitute a positive-sense single-stranded RNA genome of approximately 11.8 kb2,3. The genome encodes for five structural proteins (Capsid (CP), E3, E2, 6K and E1) and four nonstructural polyproteins (nsP1-4). Recently, CHIKV has spread widely and is the cause of a febrile illness of global concern with the potential to affect millions of people worldwide. As of 2016, Chikungunya fever has been identified in nearly 60 countries (WHO Chikungunya report; accessed 3 August 2017). Some recent outbreaks have been observed in Africa, Asia, Europe, islands in the Indian and Pacific Oceans, and recently on the Caribbean islands in America4–7. CHIKV infection is characterized by severe debilitating muscle and joint pain, and polyarthralgia, which persists for about 3–12 months and could last up to 1–3 years8–10. In some instances, severe CHIKV infection may cause neurological disorders and ocular manifestations11–13. Other symptoms include headache, myalgia, vomiting and rash14,15. Until now, there is no effective antiviral treatment, or vaccine, is commercially available for the treatment of CHIKV, and patients are treated symptomatically.\n\nStudies on antivirals for chikungunya generally target the replication machinery (nsP2 and nsP3 proteins)16–21 and surface receptors responsible for the binding of the virus during endocytosis (E1 and E2 proteins)19,21. Recent studies have shown that CHIKV is able to affect the central nervous system (CNS) like new world alphaviruses, such as Venezuelan equine encephalitis virus and Eastern equine encephalitis virus. Thus, it is important to evaluate CHIKV as a transition between new and old world viruses. Old world viruses use nsP2 to inhibit transcription of host proteins, whereas new world viruses have developed an alternative mechanism of transcription inhibition that is mainly determined by their CP protein22. Hence, CP could be an important target protein for potential antivirals. Up until now, the other structural protein of CHIKV, E3, has not been evaluated as a target for antivirals till now. E3 is the only protein in the CHIKV genome with a secretory signal.\n\nAlphavirus CP is a multifunctional protein known to act as serine protease for self-cleavage and viral genomic RNA binding. It is also known to bind to other CP molecules during nucleocapsid formation, and interact with viral spike proteins during virion formation and budding23. CHIKV CP is 261 amino acids long protein and has a molecular weight of approximately 30kDa, and contains two major domains. N-terminal domain is positively charged and is involved in non-specific RNA binding, while the C-terminal domain regulates globular protease and acts as a binding site for the spike protein24. In addition, nuclear import export signals are present on the CP’s amino acid terminal, forming immobile aggregations with nsP3 and E2 proteins of CHIKV25.\n\nThe structural protein E3 is approximately 6KDa, and is found not to be associated with the mature virion2. It serves as the signal sequence for the translocation of E3-E2-6K-E1 polyprotein into the endoplasmic reticulum, working in a clade-specific manner, and its cleavage from E2 is essential for virus maturation26. E3 also mediates pH protection of E1 during virus biogenesis via interactions strongly dependent on Y47 at the E3-E3 interface27.\n\nIn the present study, we performed an in silico analysis of protein-ligand interactions of all CHIKV proteins using a natural compound library from MolBase to predict potential antiviral compounds for CHIKV infection. Our analysis predicted three compounds that interacted with CHIKV proteins (two with the E3 protein, and one with the CP), making them potential antiviral candidates against CHIKV.\n\n\nMethods\n\nStructures of CHIKV proteins E1, E2, E3, nsP2 and nsP3 were downloaded directly from RCSB Protein Data Bank (PDB). For the rest of the CHIKV proteins, CP, 6K, nsP1 and nsP4, whose structures are unavailable, CHIKV sequences present in NCBI, belonging to ECSA (East/Central/South Africa) genotype were downloaded. These sequences were utilized to form a consensus sequence with MEGA 628 using clustalW pairwise multiple alignment algorithm with all parameters set at default. Using these consensus sequences, homologous proteins from the PDB were identified using Protein BLAST29 where the algorithm parameters were as follows: Max target sequences=100, Expect threshold=10 using BLOSUM62 scoring parameters, Gap cost=Existence:11 & Extension:1 with conditional compositional score matrix adjustment. The suitable templates for nsP1 and CP with highest query coverage, sequence identity and lowest E-value were selected for homology modeling. For proteins 6K and nsP4, no templates were available, and thus these structures were created using threading and looping method (see next section).\n\nThe template and target sequences of all CHIKV proteins were then aligned using CLUSTALW30. MODELLER (version 9.16) was used to generate homology models31. Further, the homology model having the lowest MODELLER objective function (molpdf) or DOPE or SOAP assessment scores, or the one having highest GA341 score was selected as the best model structures and were further utilized for model validation. Nonstructural protein, nsP4, and the small accessory peptide of structural protein 6K did not have any template in PDB; therefore a threading and looping approach was implemented for them using LOMETS (Local Meta Threading Server)32. Both online server and standalone program present as a module of I-TASSER Suite version 5.1, which provides 3D models by combining alignment scores of template to target of 9 different threading programs (FFAS-3D, HHsearch, MUSTER, pGenTHREADER, PPAS, PRC, PROSPECT2, SP3, and SPARKS-X). All parameters were set as default. All structural and nonstructural CHIKV protein sequences were selected as potential drug targets.\n\nGenerated models were validated using MolProbity-(v4.3.1)33. Ramachandran plot analysis was performed for the best protein models by analyzing the phi (Φ) and psi (Ѱ) torsion angles. To check reliability of the modeled structures, the root mean square deviation (RMSD) was calculated by superimposing it on template protein structure using PyMOL (v1.7.0.0) visualization software34. Consistency between templates and the modeled structures were assessed by ProSA-web35 (online server), a statistical analysis tool of all the proteins structures available at RCSB PDB. Here, a statistical average is obtained over the known structures with the help of combined potentials of mean force from the PDB database.\n\nStability of the domain regions of CHIKV protein structures was examined by molecular dynamics (MD) simulation using GROMACS (version 5.0) software package36. Optimized Potential for Liquid Simulations All-Atom37 force field was used to energy minimize the structures. Through this energy minimization, the high-energy intramolecular interactions were discarded. In order to avoid the steric clashes, overall geometry and atomic charges were also optimized. The proteins were kept at the center of the rectangular box, which was filled with SPC water model system to create the same environmental behavior of the molecules. All the atoms of the protein and the boundary of the rectangular box were separated by a minimum distance of 10 Å. 0.01M NaCl was used as a solvent exposure.\n\nThe system was further energy minimized without any restraints for 50,000-time steps; the steepest descent having step size of 0.01 ps. Then the system was equilibrated to reach a stable temperature by conducting NVT ensemble. Pressure was further equilibrated by NPT ensemble performance. The long-range electrostatic interactions were calculated by using particle mesh Ewald38 method with a cut-off of 0.9 Å for Vander Waals interactions. All the bonds were constrained by LINCS39, where only the water molecule moves to equilibrate with respect to protein structure keeping protein molecule as static. To couple the system Berendsen thermostat (V-rescale) and Parrinello-Rahman barostat were utilized to maintain the constant temperature (300 K) and pressure (1 bar). Further MD analysis was performed to observe structural changes and dynamic behavior of the protein by calculating RMSD, radius of gyration and root mean square fluctuation (RMSF) along with changes in temperature, pressure, density and total energy.\n\nSimulated computational models of CHIKV proteins were prepared and their binding sites were predicted using SiteMap (Version 2.3, 2009, Schrödinger, LLC, New York, USA). These were then used to perform molecular docking. The protein preparation wizard was used to prepare CHIKV proteins and a natural remedies library from MolBase database was prepared using the LigPrep module40. Virtual screening of modeled proteins against a natural remedy library from MolBase was done by using GLIDE module in an Extra Precision (XP) mode (Version 5.5. 2009, Schrödinger, LLC). It produces the minimal ranks of inappropriate poses and determines the appropriate binding energy of the three dimensional (3D) structure of the protein along with a ligand41,42.\n\nAfter the completion of molecular docking, the docked poses were listed depending upon the respective docking scores. Glide Score (obtained using GLIDE Module of Schrödinger Software Suite 9.0) was used as an empirical scoring function to predict free energy for ligands binding to the receptor. The structure showing minimum binding energy was filtered and subjected for further analysis. The 3D conformation ligand receptor was analyzed using PyMOL34 and Chimera43 v1.10.1 visualization software.\n\nPharmacokinetics properties and percent human oral absorption values were further predicted for the potential lead molecules using QikProp module (Version 3.2, 2009, Schrödinger, LLC)44. Both the physically remarkable descriptors and pharmaceutically admissible properties were predicted for neutralized ligands by QikProp. The program predicts 44 different properties, including log P (octanol/water), % human oral absorption in intestine (QP%) and predicted IC50 value for blockage of HERG K+ channels (log HERG). The Lipinski’s rule of five45, an important criteria for oral absorption, was evaluated for the acceptability of the compounds. In addition, admetSAR46 v1.0 was used to calculated various attributes of the drugs, including the blood brain barrier (BBB), human intestinal absorption, Caco-2 permeable, aqueous solubility, P-gp substrate and inhibitor, CYP450 substrate and inhibitor, CYP IP, ROCT, HERG inhibition, and toxicity parameters. For Lipinski score calculations, the ligand in SMILE format was uploaded to QikProp. The physicochemical properties and Lipinski Rule of Five were also analyzed by PERL script, “CalculatePhysicochemicalProperties.pl” of MayaChemTools47.\n\nThe protein-ligand complex interaction at the atomic level was analyzed using Maestro 11.0 (LLC Schrodinger 2016)48 and LigPlot+49 v1.4.5. The protein and the docked ligand were merged together and uploaded to Maestro Suite vMaestro 11. Further, the “Ligand Interaction Diagram” option was selected to draw the protein-ligand binding interactions in the 2D visualization workspace.\n\n\nResults\n\nCHIKV consists of four nonstructural proteins (nsP1-nsP4), three structural proteins (E1-E3), along with two sub-pro regions 6K and CP, which makes a part of structural protein unit (Figure 1). Structures of CHIKV proteins E1, E2, E3, nsP2 and nsP3 were downloaded directly from PDB (Figure 2a), and for other CHIKV proteins (CP, 6K, nsP1 and nspP4) homology modeling and threading and looping methods were utilized to predict their structures. For proteins with templates available, homology modeling was done with five models for every protein created based on sequence similarity using different model generation tools (MODELLER and LOMET), and validated by their internal scoring functions (molpdf, DOPE, SOAP and GA341 scores). Further, ProSA Z-score for all modeled structures were calculated to analyze the quality of models based on the Cα positions. Individual validation and ProSA Z scores for top ranked models are given in Table 1 and their structures are given in Figure 2b. The top ranked models were also analyzed by Ramachandran plot (Figure 3). The Ramachandran plot shows the distribution of phi (ϕ) and psi (ψ) angles for each amino acid residues of the modeled structures. The respective percentages of the favored and allowed regions for all the residues of all those validated are also shown in Table 1.\n\nThe genome consists of two open reading frames (ORFs) separated by an untranslated junction (J). The first ORF encodes for a polyprotein and acts as a precursor of the non-structural proteins (nsP1, nsP2, nsP3 and nsP4). The second ORF encodes the structural proteins (Capsid, E3, E2, 6K and E1). The genome has 5` cap and 3` poly A tail.\n\n(A) X-Ray structures of nsP2, E3, nsP3, E1 and E2. (B) Homology modelled structures of nsP1, Capsid, nsP4 and 6K.\n\nThis table includes validation using various simulation scores for the best ranked models for structural and nonstructural proteins of CHIKV.\n\n(A) nsP1 and Capsid (homology modeling); (B) nsP4 and 6K (Threading/Looping).\n\nMolecular dynamic simulations were employed to analyze the protein structure-function complexities, such as structural stability, conformational flexibility and folding. Domain regions of the structures (Table 2) were simulated for 20 ns. Moreover, various parameters, such as temperature, pressure, density and total energy, were calculated to check the stability of these structures along with steric properties. Graphs for temperature, pressure and density are shown in Figure 4A–C. Further, RMSD values for the backbone atoms of proteins were plotted against time of MD simulations. Radius of gyration on the other hand also supports the stability and compactness of the proteins. The RMSF with respect to each residue depicts the flexibility of the proteins. The RMSD, radius of gyration and RMSF plots are shown in Figure 4D–F. The resulting graphs contributed to protein modeling, as they show a constant RMSD deviation throughout the 20ns simulation except for a small deviation in E2 after 14ns. Depending upon these simulation parameters, the proteins showed conformational stability.\n\n(A) Temperature, (B) Pressure, (C) Density, (D) root mean square deviation (RMSD), (E) Radius of Gyration, and (F) root mean square fluctuation (RMSF). A–E graphs are vs Time, and F vs Atoms. Each set shows the graph for both Non-structural (upper) and Structural (lower) CHIKV proteins. Non-structural Protein: nsP1 (green), nsP2 (blue), nsP3 (yellow) and nsP4 (red); Structural Proteins: Capsid (orange), E3 (mustard), E2 (purple), 6K (cyan) and E1 (pink).\n\nDrug discovery relies heavily on molecular docking to understand the interactions between ligand/inhibitor and target protein50. In this study, we resorted to the docking of available protein structures (wherever applicable), as well validated, refined and simulated modeled proteins to screen against a natural remedy library from MolBase. The binding sites of all protein structures were predicted by SiteMap. The predicted binding pockets were further validated using Glide in XP mode. Top ten ligand/compounds having docking score (Glide Score) above -4, glide energy of -20 kcal mol-1 and potential energy of a considerable range were considered for the next level of screening. The combined results of all the docked ligand along with the glide energy and potential energy have been provided in Table 3. Of these, two ligands, (1,3,6, -Trigalloyl-β-D-Glucose and Quercetin-3-rutinoside (Compound ID 164 and 153) were found to interact with all the proteins and were discarded from further analysis.\n\nFor the non-structural proteins, the top ligands included Rebaudioside A and Withanoside IV (Compound ID 149 and 179) for nsP1; Stevioside, Bacopaside II and Jujubogenin isomer of bacopasaponin C (Compound ID161, 26 and 113) for nsP2; Chebulinic acid and Corilagin (Compound ID 44 and 47) for nsP3; Rebaudioside A and Stevioside (Compound ID 149 and 161) for nsP4. For structural proteins, Catechin-5-O-gallate, Rosmarinic acid and Agnuside (Compound ID 42, 151 and 18) for CP; Bacopaside II, Mangiferin and Arjungenin, (Compound ID 26, 122 and 12) for E3; (Rebaudioside A, Tribulosin and Asiaticoside (Compound ID 149, 165 and 17) for E2; Arjunetin and Stevioside (Compound ID 10 and 161) for 6K; Chebulinic acid, Stevioside and Asiaticoside (Compound ID 44, 161 and 17) for E1. Top four docked poses of the modeled proteins and the small molecules having lowest docking score are shown in Figure 5 (ligand wise).\n\nLigands are cyan sticks and receptors as pink ribbon/surface.\n\nUnique ligand-protein partners were taken forward for ADME and toxicity analysis. In case of nonstructural proteins, these ligand-protein pairs were Withanoside IV (Compound ID 179)-nsP1, Jujubogenin isomer of bacopasaponin C (Compound ID 113)-nsP2 and Corilagin (Compound ID 47)-nsP3. In case of structural proteins, these pairs were Catechin-5-O-gallate (Compound ID 42), Rosmarinic acid (Compound ID 151) and Agnuside (Compound ID 18) against CP, Mangiferin (Compound ID 122) and Arjungenin (Compound ID 12) against E3, Tribulosin (Compound ID 165) against E2, and Arjunetin (Compound ID 10) against 6K.\n\nADME screening was performed for all the top hits. Here, 44 various physically remarkable descriptors51 and pharmaceutically admissible properties of the top four lead compounds for every CHIKV protein were calculated using QikPro-P (Table 4). The Lipinski’s rule of five was further employed to evaluate oral absorption along with ADME. Compounds violating more than 2 Lipinski’s rule of 5 were discarded from further analysis.\n\nCompounds Catechin-5-O-gallate (Compound ID 42), Rosmarinic acid (Compound ID 151) and Agnuside (Compound ID 18) against CP; Mangiferin (Compound ID 122) and Arjungenin (Compound ID 12) against E3; and Arjunetin (Compound ID 10) against 6K were studied further in greater detail for their toxicity.\n\nThe efficacy and unexpected toxicity of a drug to penetrate biological barriers, such as the intestinal wall or BBB, were considered as a prime determinant of the compounds taken forwards for toxicity tests. CHIKV is an old world virus, but is now seen to affect the CNS as well; therefore, compounds that were predicted to cross the BBB were also considered as potential antivirals. Of all the compounds considered for toxicity analysis using AdmetSAR, Arjunetin (Compound ID 10) was considered ineffective for oral consumption and is also carcinogenic. Also, Agnuside (Compound ID 18) and Mangiferin (Compound ID 122) were not considered as potential antivirals as they are predicted to have positive AMES toxicity (Table 5).\n\nThe compounds that were judged to be potential antivirals were Catechin-5-O-gallate (Compound ID 42) and Rosmarinic acid (Compound ID 151) against CP and Arjungenin (Compound ID 12) against E3 structural protein of CHIKV. Thus, the ligand/drug-protein interaction was studied for these three compounds to understand their interaction pattern and strength of interaction with the protein for their role as potential antivirals against CHIKV (Table 5).\n\n+: Positive; -: Negative; NS: Non-substrate; S: Substrate; NI: Non-inhibitor; I: Inhibitors; BBB: Blood-brain barrier; CYP450: Cytochrome P450; WI: Weak inhibition; NAT: Non AMES toxic; AT: AMES toxic; NC: Non carcinogens; C: Carcinogen; HT: High toxic; RB: Readily biodegradable; NRB: Not readily biodegradable; NR: Not-required.\n\nA ligand protein interaction study was done for validated protein structures as discussed earlier. CP residues (Peptidase S3 domain) were predicted to bind to Catechin-5-O-gallate and Rosmarinic acid (Compound IDs 42 and 151, respectively) and E3 residues (Endopeptidase domain) bind to Arjungenin (Compound ID 12). The top docking conformation of Catechin-5-O-gallate showed a predicted binding energy of -6.26 kcal mol-1, whereas Rosmarinic acid and Arjungenin showed similar binding energy of -6.11 kcal mol-1 and -6.01 kcal mol-1, respectively. The binding energy (Glide Score) and the interaction energy (Potential, Vander Waals and Electrostatic) are shown in Table 3. The intermolecular hydrogen bonds and hydrophobic residues showing close contact between receptor proteins (CP and E3) and ligand (Compound ID 42, 151 and 12) are shown in Table 6 and Figure 6A–C, respectively.\n\n(A) Hydrogen bonding interaction between Catechin-5-O-gallate [CompID - 42] and capsid, binding affinity of - 6.26 kcal/mol was obtained. The zoomed region shows the receptor-binding pocket. Residues that form hydrogen bond interaction are Glu260 (Distance - 2.57 Å) and Lys 141 (Distance - 2.93 Å); His139, Val140, Asp161, Glu259 and Trp261 forms hydrophobic interaction. (B) Hydrogen bonding interaction between Rosmarinic acid [CompID - 151] and capsid, binding affinity of - 6.11 kcal/mol was obtained. The zoomed region shows the receptor-binding pocket. Residues that form hydrogen bond interaction are Glu260 (Distance - 1.71 and 1.66 Å), Trp261 (Distance - 2.04 and 1.93 Å) and Lys 141 (Distance - 2.37 and 1.99 Å); His139, Val140, Asp161 and Glu259 forms hydrophobic interaction. (C) Hydrogen bonding interaction between Arjungenin [CompID - 12] and E3, binding affinity of - 6.01 kcal/mol was obtained. The zoomed region shows the receptor-binding pocket. Residues that form hydrogen bond interaction are Gln52 (Distance - 3.11 Å) and Arg63 (Distance - 2.72 and 2.71 Å); Pro5, Ser18, Gln19, Gln49, Ala53, Ser58 and His60 forms hydrophobic interaction.\n\nThe interaction result showed that most of the hydrogen bond donors are from the protein that bind to the acceptors on the respective ligands. The compound Catechin-5-O-gallate (Compound ID 42) binds to Glu260 and Lys141 residues (HBond distance of 2.57 and 2.93 Å) of the CP protein and forms hydrophobic interactions with Asp161, His139, Val140 and Trp261 residues (Figure 7a). Further 2-D workspace revealed that when the ligand-protein interactions were observed both in the presence and absence of solvent the compound Catechin-5-O-gallate binds to the CP protein, HIS139 forms the hydrogen backbone; GLU259, GLU260, ASP161 form the hydrogen side chain. The ligand forms hydrophobic interactions with TRP261, VAL140, LYS141 (Figure 7b). We were unable to acquire the Ligplot for the interaction of Rosmarinic acid (Compound ID 151) with CP protein as the coordinates were undetectable; however, using 2-D workspace, we identified that Rosmarinic acid binds to the CP protein, TRP261 forms the hydrogen backbone; GLU260, LYS141 form the hydrogen side chain. The ligand forms hydrophobic interactions with HIS139, VAL140, GLU259, ASP161 (Figure 7b). The third compound Arjungenin (Compound ID 12), binds with Arg63 and Gln52 residues (HBond distance of 2.72 and 3.11 Å) of the E3 protein and Ser18, His60 and Ser58 residues are involved in hydrophobic interactions (Figure 7a). Its 2-D workspace revealed that SER18, GLN49 form the hydrogen backbone; GLN52, SER58, ARG63 form the hydrogen side chain. The ligand forms hydrophobic interactions with PRO5, GLN19, ALA53, HIS60 (Figure 7b). Overall docking and interaction results for the best three natural compounds have been compiled in Table 7.\n\n(A) LigPlot of Comp 42 (Capsid) and Comp 12 (E3). (B) Maestro ligand interaction diagram of Comp 42 and 151 (Capsid) and Comp 12 (E3).\n\n\nDiscussion\n\nSeveral drug candidates have been tested for their antiviral activity against CHIKV8,52,53. Recent studies have employed chemical libraries to screen for drug candidates for chikungunya with limited success16,17. Recent efforts for identifying natural compounds against alphavirus replication revealed 44 inhibitors that were effective against several alphaviruses, including CHIKV replicon and Sindbis virus. The study revealed that these compounds inhibited the early stages of viral replication19. Currently, hundreds of thousands of natural compounds are available that can be utilized for screening purposes for identifying novel drug targets. The present study was performed using virtual screening of a natural compound library from MolBase, which showed three compounds, namely, Catechin-5-O-gallate, Rosmarinic acid and Arjungenin, as promising potential antivirals against CHIKV proteins.\n\nPrevious studies have suggested that Catechin-5-O-gallate is the most important catechin in green tea, commonly known as epigallocatechin-3-gallate (EGCG). Other catechins are also found in green tea extract, such as epigallocatechin, epicatechingallate and epicatechin. The biological activity of EGCG is assumed to be contributed by the galloyl side chain54. EGCG is known to have antiviral activities towards a variety of viruses. EGCG also inhibits the cell entry of several viruses, such as human immunodeficiency virus (HIV)55–57 influenza virus58 and hepatitis C virus (HCV)59–61. Additionally, inhibitory effects of EGCG on viral transcription have been described for viruses like hepatitis B virus, adenoviruses, or herpes viruses62. In case of CHIKV, a recent study on EGCG showed inhibition of CHIKV transduction by blocking cell entry against env-pseudotyped lentiviral vectors, which inhibits CHIKV attachment63.\n\nRosmarinic acid (RA), a phenolic compound found in various Labiatae herbs64, possesses several properties, such as anti-inflammatory65,66 and antioxidative, as it reduces liver injury induced by d-galactosamine67 and lipopolysaccharides68. Besides these, RA acts as a potent antiviral agent against Japanese encephalitis virus (JEV), another alphavirus closely related to CHIKV. The study indicated that RA reduced viral replication within the brain along with the secondary inflammation resulting from microglial activation. These observations suggested that RA exhibited efficient antiviral as well as anti-inflammatory activity against Japanese equine encephalitis virus infection and hence was able to reduce disease severity66.\n\nThe compound Arjungenin, a popular triterpenoid isolated from Terminalia arjuna/ T. chebula, shows inhibitory effects on HIV-1 Protease69,70. Arjungenin has been previously used for a wide range of activities that includes anti-inflammatory, anti-microbial, anti-cancer and even anti-viral71, but no work has been done on this particular natural compound to date.\n\n\nConclusion\n\nTreatment of chikungunya includes antipyretic drugs during the febrile stage and depends heavily on symptomatic relief during the chronic arthritic phase. Our present study has identified natural compounds that may be antiviral and might be good candidates as drugs for chikungunya treatment. Further in vitro validation is required for these compounds to provide insights into their mode of action against the different stages of chikungunya infection.\n\n\nData availability\n\nAll source data relating to this article can be found in Supplementary File 1.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe authors declare that no grants were involved in supporting this work.\n\n\nSupplementary Material\n\nSupplementary File 1: Structures of CHIKV proteins (E1, E2, E3, nsP2 and nsP3) downloaded from PDB; sequences of the unknown structures found in NCBI (CP, 6K, nsP1 and nsP4); the homologous proteins identified for the unknown structure proteins in PDB and modeled structures using these template structures; protein structures prepared by threading and looping methods; the natural remedy library from MolBase and Ligand-protein docked structures.\n\nClick here to access the data.\n\n\nReferences\n\nStrauss EG, Strauss JH: Structure and replication of the alphavirus genome. 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PubMed Abstract \| Publisher Full Text\n\nFassina G, Buffa A, Benelli R, et al.: Polyphenolic antioxidant (-)-epigallocatechin-3-gallate from green tea as a candidate anti-HIV agent. AIDS. 2002; 16(6): 939–41. PubMed Abstract \| Publisher Full Text\n\nWilliamson MP, McCormick TG, Nance CL, et al.: Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy. J Allergy Clin Immunol. 2006; 118(6): 1369–74. PubMed Abstract \| Publisher Full Text\n\nKim M, Kim SY, Lee HW, et al.: Inhibition of influenza virus internalization by (-)-epigallocatechin-3-gallate. Antiviral Res. 2013; 100(2): 460–72. PubMed Abstract \| Publisher Full Text\n\nCiesek S, von Hahn T, Colpitts CC, et al.: The green tea polyphenol, epigallocatechin-3-gallate, inhibits hepatitis C virus entry. Hepatology. 2011; 54(6): 1947–55. PubMed Abstract \| Publisher Full Text\n\nCalland N, Albecka A, Belouzard S, et al.: (-)-Epigallocatechin-3-gallate is a new inhibitor of hepatitis C virus entry. Hepatology. 2012; 55(3): 720–9. PubMed Abstract \| Publisher Full Text\n\nChen C, Qiu H, Gong J, et al.: (-)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus. Arch Virol. 2012; 157(7): 1301–12. PubMed Abstract \| Publisher Full Text\n\nDas S, Tanwar J, Hameed S, et al.: Antimicrobial potential of epigallocatechin-3-gallate (EGCG): a green tea polyphenol. J Biochem Pharmacol Res. 2014; 2(3): 167–74. Reference Source\n\nWeber C, Sliva K, von Rhein C, et al.: The green tea catechin, epigallocatechin gallate inhibits chikungunya virus infection. Antiviral Res. 2015; 113: 1–3. PubMed Abstract \| Publisher Full Text\n\nShekarchi M, Hajimehdipoor H, Saeidnia S, et al.: Comparative study of rosmarinic acid content in some plants of Labiatae family. Pharmacogn Mag. 2012; 8(29): 37–41. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeake PW, Pussell BA, Martyn P, et al.: The inhibitory effect of rosmarinic acid on complement involves the C5 convertase. Int J Immunopharmacol. 1991; 13(7): 853–7. PubMed Abstract \| Publisher Full Text\n\nSwarup V, Ghosh J, Ghosh S, et al.: Antiviral and anti-inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis. Antimicrob Agents Chemother. 2007; 51(9): 3367–70. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWon J, Hur YG, Hur EM, et al.: Rosmarinic acid inhibits TCR-induced T cell activation and proliferation in an Lck-dependent manner. Eur J Immunol. 2003; 33(4): 870–9. PubMed Abstract \| Publisher Full Text\n\nPsotová J, Kolár M, Sousek J, et al.: Biological activities of Prunella vulgaris extract. Phytother Res. 2003; 17(9): 1082–7. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "25511", "date": "12 Sep 2017", "name": "Soma Chattopadhyay", "expertise": [ "Reviewer Expertise Molecular virology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present study by Jain et al entitled “In silico analysis of natural compounds targeting structural and nonstructural proteins of chikungunya virus” was undertaken to evaluate protein-ligand interactions of all chikungunya virus (CHIKV) proteins with natural compounds from a MolBase library in order to identify potential inhibitors of CHIKV. The authors have predicted three compounds, Catechin-5-O-gallate, Rosmarinic acid and Arjungenin, to interact with CHIKV proteins; two(Catechin-5-O-gallate and Rosmarinic acid) with capsid protein, and one (Arjungenin) with the E3 which required further in vitro studies to test their efficacy against CHIKV.\nThe study is designed and executed properly, however there are few points which need to be addressed before consideration.\n\nThe points are as follows:\n\nWhile considering drug like properties, authors have discarded molecules with 2 violations. Why did they not discard compounds with one violation?\n\nNo control was used in molecular docking?\n\nSince the ligands were filtered mainly based on their docking score, ligand efficiency should have been taken as the criteria for selection.\n\nFrom the final three compounds, at least catechin-5-O-gallate and rosmarinic acid are associated with poor absolute oral bioavailability. Then how they can be advanced as drug candidates?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3227", "date": "08 Dec 2017", "name": "Sujatha Sunil", "role": "Author Response", "response": "We thank the reviewer for her valuable comments and suggestions. Point by point replies to their queries are mentioned below. While considering drug like properties, authors have discarded molecules with 2 violations. Why did they not discard compounds with one violation? Answer: One violation (20% violation) was kept as it showed better results for toxicity and thus was allowed. No control was used in molecular docking? Answer: Docking is an insilico process and defined by parameters and not controls. Thus, no control was taken for the study. Since the ligands were filtered mainly based on their docking score, ligand efficiency should have been taken as the criteria for selection. Answer: Ligand efficiency is calculated by the binding energy as well as h-bonds of ligand and protein and is also included in the results. From the final three compounds, at least catechin-5-O-gallate and rosmarinic acid are associated with poor absolute oral bioavailability. Then how they can be advanced as drug candidates? Answer: All these compounds were positive for human intestinal absorption for oral bioavailability based on our in silico toxicity assays and thus could be advanced as drug candidates if we pursue the studies. However, to avoid confusion based on Caco permeability data that showed negative for these compounds, we have removed this data from the revised manuscript." } ] }, { "id": "27576", "date": "02 Nov 2017", "name": "Debasis Nayak", "expertise": [ "Reviewer Expertise Virology", "immunology", "imaging" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall comment: The article in the present form needs some revision including through checking of grammar and sentence structure. Further, I would request the authors to consider following points.\n\nAuthors have written that \"All bonds were constrained by LINCS\". This claim needs to revisit. Generally, bonds involving hydrogen atoms are restrained using LINCS.\n\nAuthors have mentioned that \"In order to avoid the steric clashes, overall geometry and atomic charges were also optimized\". I do not think authors have optimized the atomic charges. Rather atomic charges were obtained from the OPLS forcefield that was used in the simulations.\n\nThe cutoff used for PME was 0.9 Angstrom. This is very small. It should rather be 9 Angstrom.\n\nBerendsen thermostat with V-rescale is not a good option for conformational sampling. A better option is Langevin dynamics.\n\nAuthors have written that \"The system was further energy minimized without any restraints for 50,000-time steps; the steepest descent having step size 0f 0.01 ps\". This claim needs to revisit. No time-step is required for energy minimization. Only to study dynamics, i.e, to integrate Newton's equation of motion, we need to set time-step.\n\nIt was not mentioned what time-step was used for the heating stage and production simulation.\n\nDocking is not a good method for predicting the binding free energy. It is the least accurate method for estimating the binding free energy. However, it is a very good method for predicting the binding pose. Authors should have conducted MM-PBSA type analysis to estimate the binding free energy.\n\nFigure 4 A, B, C are not required.\n\nLooking at Figure 4D, one can see that the for a couple of cases, longer simulation is required. The simulation length is too small (20 ns) to investigate the dynamics of proteins. They should extend all simulations to 50 ns.\n\nFigure 4: Use bigger fonts. It's not readable.\n\nCalculate average RMSD and Radius of Gyration during the simulations.\n\nAuthors have written that \"RMSF with respect to each residue....\". RMSF is calculated with respect to the average structure obtained from simulations. Do you use C-alpha atoms of each residue?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3226", "date": "08 Dec 2017", "name": "Sujatha Sunil", "role": "Author Response", "response": "We thank the reviewers for their valuable comments and suggestions. We have considered all their suggestions and have incorporated them in the updated version of the manuscript. Point by point replies to their queries are mentioned below. Authors have written that \"All bonds were constrained by LINCS\". This claim needs to revisit. Generally, bonds involving hydrogen atoms are restrained using LINCS. Answer: Depending on the time-step applied, setting a constraint to bonds is required. The time-step depends on the highest frequency of motion within the system. If a time-step superior to 1 fs is chosen (as in our study, we have chosen 2 fs time-step), bonds involving hydrogen will not be sampled sufficiently, thus they should be constrained. Here, we have constrained all-bonds even heavy atom-H bonds by LINCS algorithm. Authors have mentioned that \"In order to avoid the steric clashes, overall geometry and atomic charges were also optimized\". I do not think authors have optimized the atomic charges. Rather atomic charges were obtained from the OPLS forcefield that was used in the simulations. Answer: The initial structure of the protein in optimized and the atomic charges of the protein are calculated by using the OPLS force field. The cutoff used for PME was 0.9 Angstrom. This is very small. It should rather be 9 Angstrom. Answer: We apologize for the confusion. The cutoff used for PME was 0.9 nm, Angstrom has been replaced by nm in the revised MS. Berendsen thermostat with V-rescale is not a good option for conformational sampling. A better option is Langevin dynamics. Answer: Berendsen thermostat was used to re-scale the velocities of particles in molecular dynamics simulations to control the simulation temperature. No conformational sampling was done in the study. Authors have written that \"The system was further energy minimized without any restraints for 50,000-time steps; the steepest descent having step size 0f 0.01 ps\". This claim needs to revisit. No time-step is required for energy minimization. Only to study dynamics, i.e, to integrate Newton's equation of motion, we need to set time-step. Answer: Time step (nsteps) of 50000 i.e the maximum number of (minimization) steps to perform was provided during energy minimization. Below are the parameters provided in mdp file. ; minim.mdp - used as input into grompp to generate em.tpr ; Parameters describing what to do, when to stop and what to save integrator = steep ; Algorithm (steep = steepest descent minimization) emtol = 1000.0 ; Stop minimization when the maximum force < 1000.0 kJ/mol/nm emstep = 0.01 ; Energy step size nsteps = 50000 Maximum number of (minimization) steps to perform ; Parameters describing how to find the neighbors of each atom and how to calculate the interactions nstlist = 1 ; Frequency to update the neighbor list and long range forces ns_type = grid ; Method to determine neighbor list (simple, grid) rlist = 1.0 ; Cut-off for making neighbor list (short range forces) coulombtype = PME ; Treatment of long range electrostatic interactions rcoulomb = 1.0 ; Short-range electrostatic cut-off rvdw = 1.0 ; Short-range Van der Waals cut-off pbc = xyz ; Periodic Boundary Conditions (yes/no) It was not mentioned what time-step was used for the heating stage and production simulation. Answer: Time-step of 20 ns was used for production simulations. Below is the parameter details provided in mdp file for production simulations. nsteps = 10000000; 2 * 10000000 = 20000 ps , 20 ns Docking is not a good method for predicting the binding free energy. It is the least accurate method for estimating the binding free energy. However, it is a very good method for predicting the binding pose. Authors should have conducted MM-PBSA type analysis to estimate the binding free energy. Answer: The study was to screen the natural compounds against CHIKV structural and non-structural proteins and identify potential inhibitors. MM-PBSA for a large number of compounds was out of the scope of the present study and hence not pursued. Figure 4 A, B, C are not required. Answer: We abide by the reviewer’s recommendation. The figures have been removed and changes figure is attached to the revised manuscript. Looking at Figure 4D, one can see that the for a couple of cases, longer simulation is required. The simulation length is too small (20 ns) to investigate the dynamics of proteins. They should extend all simulations to 50 ns. Answer: Except for E2, every simulation has reached to a stable state as shown in the RMSD graph. Figure 4: Use bigger fonts. It's not readable. Answer: We apologize for this inconvenience. The figure has been changed. Calculate average RMSD and Radius of Gyration during the simulations. Answer: RMSD Average - 22.93 Radius of Gyration Average - 1.45 Authors have written that \"RMSF with respect to each residue....\". RMSF is calculated with respect to the average structure obtained from simulations. Do you use C-alpha atoms of each residue? Answer: As one can see in graph 4F, the x-axis represents the atomic positions in the trajectory after fitting to a reference frame. Yes, C-alpha atoms of each residue have been used for the analysis." } ] } ]	1	https://f1000research.com/articles/6-1601
https://f1000research.com/articles/6-2103/v1	07 Dec 17	{ "type": "Research Article", "title": "Evaluation of antibiotic susceptibility in wound infections: A pilot study from Bangladesh", "authors": [ "Sushmita Roy", "Mejbah Uddin Ahmed", "Bhuiyan Mohammad Mahtab Uddin", "Zubair Ahmed Ratan", "Monali Rajawat", "Varshil Mehta", "Sojib Bin Zaman", "Sushmita Roy", "Mejbah Uddin Ahmed", "Bhuiyan Mohammad Mahtab Uddin", "Zubair Ahmed Ratan", "Monali Rajawat", "Varshil Mehta" ], "abstract": "Introduction: Infections due to antibiotic resistant bacteria have increased alarmingly in both developed and developing countries. Unrestrained and rapidly spreading bacterial growth has turned the management of wound infections into a serious challenge. This study aimed to determine the prevalence of different bacterial pathogens and their antibiotic susceptibility in various types of wound infections. Methods: A cross-sectional study was conducted to collect 105 wound swabs. All isolated bacteria were identified based on colony characteristics, gram stain and standard biochemical tests, and antibiotic susceptibility testing (AST) with the disc diffusion method. Descriptive statistics were used to present the study findings, and all analyses were performed using Stata Version 13. Results: The rate of isolation of bacteria was 92.3%. Staphylococcus aureus was found to be the most frequent isolate (55.7%), followed by Escherichia coli (23.7%), Pseudomonas spp. (8.2%), and Streptococcus pyogenes (7.2%). Gram-positive bacteria were mostly (60%) found sensitive to vancomycin, azithromycin, gentamicin, imipenem, cefixime, and ceftriaxone in this study. Among the Gram-negative bacteria, Escherichia coli (>60%) showed sensitivity to cefixime, azithromycin, cefuroxime, ceftriaxone, cefotaxime, gentamycin, and ceftazidime. Conclusions: The diversity of isolated bacteria and their susceptibility patterns signify a need to implement a proper infection control strategy, which can be achieved by carrying out antibiotic sensitivity tests of the isolates.", "keywords": [ "wound infection", "bacterial pathogen", "antibiotic susceptibility pattern" ], "content": "Introduction\n\nWounds follow the loss of skin integrity, which provides a moist, warm and nutritive environment that is known to be conducive to microbial colonization and proliferation1. Wound infections are considered a major complication of surgery, and can be classified into three types: incisional surgical wounds, deep incisional wounds, and organ-specific infections2. Despite maintaining the high standards of preoperative preparation, antibiotic prophylaxis, and operative procedures, the appearance of postoperative wound infections remains a grave threat among the clinicians3. Some of the most frequent causative microorganisms are related to wound infections and include Staphylococcus aureus, Streptococcus pyogenes, Enterococci, Escherichia coli, Klebsiella pneumonia, Proteus species and Pseudomonas aeruginosa. However, the severity of complication is largely based on the virulence of the infecting pathogen and the site of infection4. The reporting trend of infection varies depending on the surgeon’s ability, operative area, surgical procedures, patient characteristics, etc. For instance, approximately 5,00,000 infections per year take place in the United States among an estimated 27 million surgical procedures5. The incidence of hospital-based postoperative infection varies from 10%–25% in India6. Nosocomial infection is becoming a serious problem affecting hospitalized patients both in developed and developing countries. According to a study conducted in Bangladesh, it was reported that among 38% of nosocomial infections, more than 50% were due to wound infection7. Moreover, wound infections were found to be higher (49%) among post-operative patients as compared to pre-operative patients (15.9%) in that study7. Post-operative wound infections have emerged as one of the important causes of morbidity among the hospitalized patients8. Emmerson et al. reported that surgical wound infections account for 12.3% of all hospital-acquired infections9. Wound infection is becoming a major concern among patients and healthcare practitioners for its increased toll on morbidity and financial loss. It also generates demand for attaining expensive management within the public health system5. Active and passive surveillance of surgical site infections in the hospital will help the surgeons and clinicians to know the antibiotic susceptibility pattern related to the surgical site, which can help reduce postoperative complications10.\n\nThe present study aimed to collect data on the bacteriological profiles of wound infections and their antibiotic susceptibility patterns in a teaching hospital in Bangladesh.\n\n\nMethods\n\nThis cross-sectional study was conducted from the 10th of July 2016 to the 30th December 2016.\n\n105 samples of pus or wound swab were collected from the Microbiology Department of the Enam Medical College Hospital, Dhaka, which is a teaching hospital located in Bangladesh. The Microbiology department collected the samples from the outpatient and inpatient department of Surgery, Medicine, Gynaecology, and Orthopaedic.\n\n105 swab samples were collected from patients with various wound infections including post-operative surgical wounds, burn wounds and superficial and soft tissue infections (SSTI) by paramedics. Selective criteria were considered: infected wound, adult patients, and before administration of antibiotics. Specimens were collected aseptically by nurses or technicians before the wound cleaning and before application of an antiseptic solution. At the time of swab collection, standard care was taken to avoid contamination by the normal flora of the surrounding skin. Then the specimens were transported within one hour to the Microbiology laboratory of the hospital to perform the culture and susceptibility tests. Subsequently, each specimen was inoculated on appropriate agar media: blood agar, MacConkey’s agar, nutrient agar, and mannitol salt agar media. Finally, the cultures were incubated aerobically at 37°C for 24–48 hours with proper care. All the plates were regularly inspected for growth, and identification of the isolated bacteria was done by colony morphology, gram-staining and standard biochemical tests by microbiologists11. Antimicrobial susceptibility patterns of the isolated bacterial pathogens were tested by using commonly used antibiotics such as amoxicillin (10 µg), penicillin (10 µg), vancomycin (30 µg), azithromycin (15 µg), cephradine (30 µg), tetracycline (30 µg), cloxacillin (5 µg), co-trimoxazole (23.75 µg), ciprofloxacin (5 µg), cefixime (5 µg), cefuroxime (30 µg), imipenem (10 µg), ceftriaxone (30 µg), and nitrofurantoin (300 µg) using the Kirby Bauer disc diffusion method according to the guidelines of Clinical Laboratory Standards Institute12.\n\nErrors in data were revised after cross-checking the laboratory records and clinical case recording forms. Descriptive statistics were used to interpret the data. Frequency and proportions were used to present categorical variables while mean and standard deviation (SD) were given to describe continuous variables. Stata (v.13) was utilized to analyze the data.\n\nWritten informed consent was obtained from each participant. All study participants were informed verbally about the objective of the study. The research team paid the costs related to patient sample collection. The study was conducted under the clearance of the Ethical Review Committee (approval# 2017/218) of Enam Medical College Hospital, Dhaka, Bangladesh.\n\n\nResults\n\nThe mean (±standard deviation) age of the study participants was 37 (±08) years, and 57.1% of participants were male. The rate of isolation of bacteria was 92.3%. Figure 1 shows the frequency of bacterial growth. Around 62.9% of culture positive plates turned out to be Gram-positive organisms, and 37.1% Gram-negative (n=97). Only 7.6% did not yield any growth in a culture plate.\n\nIn this figure, red, magenta, and green portion indicates the Gram Positive, Gram Negative, No growth, respectively and indicates the percentage of bacterial growth.\n\nStaphylococcus aureus (n=54; 55.7%) was predominantly found to be isolated among all the presenting bacteria. The frequency of Escherichia coli and Pseudomonas species was 23.7% and 8.2%, respectively (Figure 2).\n\nRate of isolation of different bacteria are mentioned here based on number and their corresponding percentage.\n\nThe susceptibility pattern of Gram-positive bacteria was mostly isolated to imipenem (90%), followed by ceftriaxone (85.5%), gentamycin (81.8%), vancomycin (80.8%), azithromycin (76.5%) and other antibiotics (<75.0%) (Table 1).\n\nMost of the Gram-negative isolates were sensitive to ceftazidime (79.0%), ceftriaxone (71.8%), gentamicin (70.7%) and other antibiotics (<70.0%) (Table 2). Most of the Pseudomonas spp. (>50%) were sensitive to ceftriaxone, imipenem, and gentamycin.\n\n\nDiscussion\n\nManagement of post-operative wound infection remains a significant concern for physicians globally13. The problem has magnified due to the rapidly spreading resistance to the available array of antimicrobial agents14,15. We found that Gram-positive organisms accounted for 62.9% of isolates, compared to Gram-negative isolates that accounted for 37.1%. Staphylococcus aureus (55.7%) was the major microbial pathogen responsible for the wound infections. According to Centre for Disease Control and Prevention (CDC), Staphylococcus aureus is the most common organism associated with surgical wound infections. This study supports the results reported by Nwachukwu et al.16, where 42.3% of infections were found to be caused by Staphylococcus aureus. Among the Gram-negative organisms, Escherichia coli were frequently isolated (23.7%) in our study. This finding is in line with a previous study which identified Escherichia coli as the major pathogen in the wound infection, followed by Staphylococcus aureus in a different setup17. A previous survey conducted in Lahore supported our findings demonstrating that Staphylococcus aureus was the main causative organism of surgical infection18.\n\nIn our study, we found imipenem as the most active antibiotic, with a susceptibility of 94.4% against Staphylococcus aureus. This study showed high sensitivity of Staphylococcus aureus against imipenem, vancomycin, and gentamycin. This finding corresponds to a previous study that also found that Staphylococcus aureus was susceptible to higher generation of antibiotics19. The high sensitivity to gentamycin has also been reported by other authors as well20. We found that Staphylococcus aureus is usually resistant to various antibiotics and the infection might be acquired in the hospital.\n\nAmong the Gram-negative bacteria, Escherichia coli was found to be susceptible to ceftriaxone, cefotaxime, gentamycin, cefixime, ceftazidime, and cefuroxime. Furthermore, we found that Escherichia coli were less sensitive to cloxacillin with a frequency of 47.8%. Among three isolated Klebsiella spp., all organisms were resistant to cephradine, penicillin, cloxacillin, cefuroxime, tetracycline, and ciprofloxacin. Similarly, Okonko et al.21 had observed a high level of resistance by Klebsiella spp. to most antibiotics. However, they noticed that all three Klebsiella spp. Isolates were susceptible to gentamycin and ceftazidime. This high susceptibility pattern might support gentamycin as a suitable antibiotics to treat Klebsiella infection22. Among eight isolated Pseudomonas spp., all were resistant to cephradine, penicillin, cloxacillin, cefuroxime, amoxicillin, and cefotaxime in this study. We found that five Pseudomonas isolates were susceptible to ceftriaxone, four were susceptible to imipenem and gentamycin, and three were susceptible to tetracycline, ciprofloxacin, azithromycin, and ceftazidime. Only one Pseudomonas spp. isolate was susceptible to co-trimoxazole, cefixime, and nitrofurantoin.\n\nThe susceptibility pattern that we found indicates that most of the isolated strains were multi-drug resistant. Similarly, a study conducted in European setting reported a high resistance of Pseudomonas spp., mostly isolated from surgical wounds23. Several previous studies carried out in different settings also support the multi-drug resistance pattern of Pseudomonas spp.24–26. The mechanisms of intrinsic resistance of Pseudomonas spp. over most of the antimicrobial agents has emerged because of the low permeability of its outer membrane and the naturally occurring chromosomal Amp β-lactamase27,28.\n\nThe control of wound infections is becoming difficult due to widespread bacterial resistance to antibiotics. Previous studies also notified an increased incidence of bacterial infections by methicillin-resistant Staphylococcus aureus, polymicrobial flora and different fungi29. As wound infections are found to be common in this study, prior knowledge of the causative agents of can be a helpful tool in selecting the empiric antimicrobial therapy to control infection\n\nIn developing countries, physicians generally do not wait for the culture reports and sometimes, there may be a delay in conducting or reporting of a culture sensitivity test. Hence, with our study, we would like to urge the physicians to start an empirical therapy with a combination of either of the following as an empirical treatment regime:\n\n1) Azithromycin/Imipenem and Ceftriaxone;\n\n2) Gentamycin and Imipenem/Ceftriaxone\n\n3) Ceftazidime and Imipenem.\n\nAfter application of the above mentioned combination regime, culture sensitivity is advised to be performed in next step. Irrespective of the report, the entire course should be completed and if the condition still remains has not improved, urgent change of treatment plan according to the culture sensitivity report should be carried out.\n\nWe would discourage the use of penicillin and amoxicillin, since the resistance towards them has been on the rise. We would also urge physicians to not to prescribe the last resort drugs like vancomycin and linezolid, since they should be used as only in high resistance cases.\n\nThe susceptibility patterns of bacterial isolates to commonly prescribed antibiotics like ceftriaxone, cefuroxime, ciprofloxacin, and azithromycin might not be generalized globally. The fact that our research was a single center study and had a small sample size were other drawbacks. However, our result might represent the scenario of a developing country. Moreover, high-quality data and laboratory support were the particular strengths of this study.\n\n\nConclusions\n\nThe most common isolate in wound infection was Staphylococcus aureus, followed by Escherichia coli, Pseudomonas spp., Klebsiella spp., and Streptococcus pyogenes. Gram-negative bacteria were sensitive to fewer than thirty percent of the commonly prescribed antibiotics, which can be a matter of great concern when treating wound infections. The judicious use of antibiotic prophylaxis and reporting can be the most effective means to reduce the wound infection rate.\n\n\nData availability\n\nDataset 1: Patient characteristics. Age, sex, residence, occupation, blood pressure, and diabetic mellitus status are given. DOI, 10.5256/f1000research.12887.d18574030.\n\nDataset 2: Antibiotic susceptibility of bacterial cultures. Includes data on S. aureus, S. pyogenes, E. coli, Klebsiella spp, Pseudomonas, and Proteus. DOI, 10.5256/f1000research.12887.d18574131.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe are grateful to the director of the hospital for giving us the permission to collect data.\n\n\nReferences\n\nInsan NG, Payal N, Singh M, et al.: Post operative wound infection: Bacteriology and antibiotic sensitivity pattern. International Journal of Current Research and Review. 2013; 5(13): 74–79. Reference Source\n\nHoward R, Lee J: Surgical wound infections: epidemiology, surveillance, and clinical management. Surgical Infectious Diseases. 1995; 401–12.\n\nBowler PG, Duerden BI, Armstrong DG: Wound microbiology and associated approaches to wound management. Clin Microbiol Rev. 2001; 14(2): 244–69. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLilani S, Jangale N, Chowdhary A, et al.: Surgical site infection in clean and clean-contaminated cases. Indian J Med Microbiol. 2005; 23(4): 249–52. PubMed Abstract\n\nHaley RW, Culver DH, White JW, et al.: The nationwide nosocomial infection rate. A new need for vital statistics. Am J Epidemiol. 1985; 121(2): 159–67. PubMed Abstract \| Publisher Full Text\n\nMustafa A: Incidence of nosocomial wound infection in postoperative patients at a teaching hospital in Kashmir. JK— Practitioner. 2004; 2(1): 38–4.\n\nHussain T, Fazal M, Ahmed A: Nosocomial infection-A cross-sectional study in the surgical wards of Dhaka Medical College Hospital. J Prev Soc Med. 1991; 10: 70–3.\n\nKoontz FP: Trends in post-operative infections by Gram-positive bacteria. Int J Antimicrob Agents. 2000; 16 Suppl 1: S35–7. PubMed Abstract \| Publisher Full Text\n\nEmmerson AM, Enstone JE, Griffin M, et al.: The Second National Prevalence Survey of infection in hospitals--overview of the results. J Hosp Infect. 1996; 32(3): 175–90. PubMed Abstract \| Publisher Full Text\n\nZaman SB, Hussain MA, Nye R, et al.: A Review on Antibiotic Resistance: Alarm Bells are Ringing. Cureus. 2017; 9(6): e1403. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCheesbrough M: District laboratory practice in tropical countries. Cambridge university press; 2006. Reference Source\n\nCLSI C: Performance Standards for Antimicrobial Susceptibility Testing: Twentieth Informational Supplement. CLSI Document M100-S20, Clinical and Laboratory Standards Institute, Wayne, Pa USA; 2010; 30.\n\nZaman SB, Hussain MA, Hossain N, et al.: Antibiotic Resistance: A Tragedy of the Common. International Journal of Research Studies. 2017; 1(2): 7–9. Reference Source\n\nRaza MS, Chander A, Ranabhat A: Antimicrobial susceptibility patterns of the bacterial isolates in post-operative wound infections in a tertiary care hospital, Kathmandu, Nepal. Open Journal of Medical Microbiology. 2013; 3(3): 159. Publisher Full Text\n\nDionigi R, Rovera F, Dionigi G, et al.: Risk factors in surgery. J Chemother. 2001; 13(Spec No 1(1)): 6–11. PubMed Abstract \| Publisher Full Text\n\nNwachukwu NC, Orji FA, Okike UM: Antibiotic susceptibility patterns of bacterial isolates from surgical wounds in Abia State University Teaching Hospital (ABSUTH), Aba–Nigeria. Research Journal of Medicine and Medical Sciences. 2009; 4(2): 575–9. Reference Source\n\nAfroz H, Fakruddin M, Masud MR, et al.: Incidence of and risk factors for Hospital Acquired Infection in a Tertiary Care Hospital of Dhaka, Bangladesh. Bangladesh Journal of Medical Science. 2017; 16(3): 358–69. Publisher Full Text\n\nAman S: Bacteriological analysis of wound infection in Mayo hospital, Lahore. J Pak Med Assoc. 1982; 32(3): 66–68. PubMed Abstract\n\nMengesha RE, Kasa BG, Saravanan M, et al.: Aerobic bacteria in post surgical wound infections and pattern of their antimicrobial susceptibility in Ayder Teaching and Referral Hospital, Mekelle, Ethiopia. BMC Res Notes. 2014; 7(1): 575. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZaman SB, Hossain N, Yasir Arafat SM, et al.: Management of Newborn Infection: Knowledge and attitude among health care providers of selected sub-district hospitals in Bangladesh. International Journal of Perceptions in Public Health. 2017; 1(2): 127–32. Reference Source\n\nOkonko IO, Soleye FA, Amusan TA, et al.: Incidence of multi-drug resistance (MDR) organisms in Abeokuta, Southwestern Nigeria. Global Journal of Pharmacology. 2009; 3(2): 69–80. Reference Source\n\nAbe-Aibinu IE, Ohaegbulam V, Odugbemi TO: A comparative study on the antimicrobial susceptibility patterns of Klebsiella and Enterobacter species from the Lagos university teaching hospital. Journal of the Nigerian Infection Control Association. 2000; 3(2): 14–7. Publisher Full Text\n\nFluit AC, Jones ME, Schmitz FJ, et al.: Antimicrobial susceptibility and frequency of occurrence of clinical blood isolates in Europe from the SENTRY antimicrobial surveillance proGram, 1997 and 1998. Clin Infect Dis. 2000; 30(3): 454–60. PubMed Abstract \| Publisher Full Text\n\nDaini OA, Effiong MJ, Ogbolu OD: Quinolones Resistance and R-Plasmids of clinical isolates of Pseudomonas species. Sudan JM Sci. 2008; 3(2): 139–46. Publisher Full Text\n\nSekiguchi J, Asagi T, Miyoshi-Akiyama T, et al.: Multidrug-resistant Pseudomonas aeruginosa strain that caused an outbreak in a neurosurgery ward and its aac(6')-Iae gene cassette encoding a novel aminoglycoside acetyltransferase. Antimicrob Agents Chemother. 2005; 49(9): 3734–42. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNordmann P, Guibert M: Extended-spectrum beta-lactamases in Pseudomonas aeruginosa. J Antimicrob Chemother. 1998; 42(2): 128–31. PubMed Abstract\n\nSexton DJ: The impact of antimicrobial resistance on empiric antibiotic selection and antimicrobial use in clinical practice. J Med Liban. 2000; 48(4): 215–20. PubMed Abstract\n\nOlayinka AT, Olayinka BO, Onile BA: Antibiotic susceptibility and plasmid pattern of Pseudomonas aeruginosa from the surgical unit of a university teaching hospital in north central Nigeria. International Journal of Medicine and Medical Sciences. 2009; 1(3): 079–83. Reference Source\n\nShittu AO, Kolawole DO, Oyedepo EA: A study of wound infections in two health institutions in Ile-Ife, Nigeria. Afr J Biomed Res. 2002; 5(3): 97–102. Publisher Full Text\n\nRoy S, Ahmed MU, Uddin BMM, et al.: Dataset 1 in: Evaluation of antibiotic susceptibility in wound infections: A pilot study from Bangladesh. F1000Research. 2017. Data Source\n\nRoy S, Ahmed MU, Uddin BMM, et al.: Dataset 2 in: Evaluation of antibiotic susceptibility in wound infections: A pilot study from Bangladesh. F1000Research. 2017. Data Source" }	[ { "id": "29451", "date": "22 Jan 2018", "name": "Md. Abu Sayeed", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAuthors studied the prevalence of different bacterial pathogens and their antibiotic susceptibility in various types of wound infections. They used 105 samples. They studied colony morphology, and performed Gram-staining and standard biochemical tests to identify the bacteria. Authors used the disc diffusion method for assessing antibiotic susceptibility. Authors found 62.9% Gram-positive and 37.1% Gram-negative bacteria in their samples.They found Staphylococcus aureus (55.7%), Escherichia coli (23.7%), Pseudomonas spp. (8.2%), Streptococcus pyogenes (7.2%), Klebsiella spp. (3.1%), and Proteus spp. (2.1%). Authors stated that Staphylococcus aureus (>50%) was sensitive to all antibiotics they used, while Streptococcus pyogenes (>50%) was sensitive to all antibiotics except co-trimoxazole. Escherichia coli (>50%) was sensitive to co-trimoxazole, cefixime, aztreonam, cefuroxime, tetracycline, imipenem, ceftriaxone, cefotaxime, gentamycin, ceftazidime, nitrofurantoin; Klebsiella sp. (>60%) was sensitive to azithromycin, gentamycin, ceftazidime; Pseudomonas sp. (>50%) was sensitive to imipenem, ceftriaxone, gentamycin; Proteus sp. (>50%) was sensitive to all antibiotics except cephradine, penicillin, cloxacillin, amoxicillin.\n\nThis manuscript is suitable for indexing after minor revision. My suggestions are here:\n\n-English needs revision.\n\nAbstract section -Methods: The headline of this paragraph should be Materials and Methods.\n-Results: This section seems incomprehensive. For example, authors stated the percentage of four isolates only where they found six, authors should also include the other two isolates. Authors only stated the sensitivity of gram-positive bacteria to different antibiotics; authors should also state the sensitivity of gram-negative bacteria.\n-Conclusions: based on the study objectives, what is the significance of the study?\n\nIntroduction section -Lines 11-14: should provide reference. -Lines 14-15: need more appropriate reference. -Lines 18-20: should provide recent information and the reference. -Lines 31-32: surgical wound infections rate should be corrected based on the cited reference.\n\nMethods section -The headline of this paragraph should be Materials and Methods.\n-Data collection: Did authors use the ‘commercially prepared paper disc’ of all the antibiotics or prepare in their laboratory? Authors should mention the company name of all antibiotics. Authors should mention which biochemical tests they performed.\n\nResults section -Isolation of different types of bacteria: should mention the information of all isolated bacteria.\n-Isolation of different types of bacteria and Sensitivity pattern of isolated Gram-positive and Gram-negative bacteria: if authors would include some photographs of ‘biochemical tests’ and ‘sensitivity tests’ that would definitely increase the strength of the article, even without photographs the manuscript is ok.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3393", "date": "02 Feb 2018", "name": "Varshil Mehta", "role": "Author Response", "response": "Thank you for your comments and expert opinion. We will certainly update the article as per your suggestions." }, { "c_id": "3450", "date": "21 Feb 2018", "name": "Sojib Bin Zaman", "role": "Author Response", "response": "Many thanks for sending us the review and expert judgement. Highly thoughtful. Agree with most of your concern. We will update the revised version addressing your comments." } ] }, { "id": "30324", "date": "19 Feb 2018", "name": "Mohammad Siddiqur Rahman Khan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is suitable to be indexed after some minor corrections.\n\nMethods: The title should be Materials and Methods.\nIntroduction: The source of infection was not clear from the description. That will be a valuable information and help others to minimize the hospital-acquired infection.\n\nData collection: Specimen transportation temperature was not mentioned.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3449", "date": "21 Feb 2018", "name": "Sojib Bin Zaman", "role": "Author Response", "response": "I want to thank you for sending us your review and expert opinion. We will certainly address all the comments point by point in the updated version. We really appreciated your valuable time and effort." } ] } ]	1	https://f1000research.com/articles/6-2103
https://f1000research.com/articles/6-1843/v1	16 Oct 17	{ "type": "Opinion Article", "title": "Developing data interoperability using standards: A wheat community use case", "authors": [ "Esther Dzale Yeumo", "Michael Alaux", "Elizabeth Arnaud", "Sophie Aubin", "Ute Baumann", "Patrice Buche", "Laurel Cooper", "Hanna Ćwiek-Kupczyńska", "Robert P. Davey", "Richard Allan Fulss", "Clement Jonquet", "Marie-Angélique Laporte", "Pierre Larmande", "Cyril Pommier", "Vassilis Protonotarios", "Carmen Reverte", "Rosemary Shrestha", "Imma Subirats", "Aravind Venkatesan", "Alex Whan", "Hadi Quesneville", "Esther Dzale Yeumo", "Michael Alaux", "Elizabeth Arnaud", "Sophie Aubin", "Ute Baumann", "Patrice Buche", "Laurel Cooper", "Hanna Ćwiek-Kupczyńska", "Robert P. Davey", "Richard Allan Fulss", "Clement Jonquet", "Marie-Angélique Laporte", "Pierre Larmande", "Cyril Pommier", "Vassilis Protonotarios", "Carmen Reverte", "Rosemary Shrestha", "Imma Subirats", "Aravind Venkatesan", "Alex Whan" ], "abstract": "In this article, we present a joint effort of the wheat research community, along with data and ontology experts, to develop wheat data interoperability guidelines. Interoperability is the ability of two or more systems and devices to cooperate and exchange data, and interpret that shared information. Interoperability is a growing concern to the wheat scientific community, and agriculture in general, as the need to interpret the deluge of data obtained through high-throughput technologies grows. Agreeing on common data formats, metadata, and vocabulary standards is an important step to obtain the required data interoperability level in order to add value by encouraging data sharing, and subsequently facilitate the extraction of new information from existing and new datasets. During a period of more than 18 months, the RDA Wheat Data Interoperability Working Group (WDI-WG) surveyed the wheat research community about the use of data standards, then discussed and selected a set of recommendations based on consensual criteria. The recommendations promote standards for data types identified by the wheat research community as the most important for the coming years: nucleotide sequence variants, genome annotations, phenotypes, germplasm data, gene expression experiments, and physical maps. For each of these data types, the guidelines recommend best practices in terms of use of data formats, metadata standards and ontologies. In addition to the best practices, the guidelines provide examples of tools and implementations that are likely to facilitate the adoption of the recommendations. To maximize the adoption of the recommendations, the WDI-WG used a community-driven approach that involved the wheat research community from the start, took into account their needs and practices, and provided them with a framework to keep the recommendations up to date. We also report this approach’s potential to be generalizable to other (agricultural) domains.", "keywords": [ "wheat", "data interoperability", "metadata", "ontology repository", "bio-ontologies", "standard vocabularies", "data formats" ], "content": "Introduction\n\nWheat was one of the first domesticated food crops, and for 8000 years it has been the basic staple food of major civilizations in Europe, West Asia, and North Africa. According to the International Wheat Initiative (IWI, http://www.wheatinitiative.org/), a framework to establish strategic research and organization priorities for wheat research at the international level in both developed and developing countries, wheat is the most widely grown cereal grain, cultivated in about 17% of the total arable land globally, and the staple food for 35% of the world’s population, providing 20% of all calories consumed by people worldwide and more protein in the human diet than any other crop (http://www.wheatinitiative.org./about-wheat/factsheets-infographics). According to the Consultative Group on International Agricultural Research’s research program on Wheat (http://wheat.org), an estimated 1.2 billion poor people depend on wheat, a crop that is particularly vulnerable to climate change.\n\nThe IWI has identified easy access and interoperability of all wheat-related data as a top priority for the wheat research community, which is in line with FAIR data principles1. Interoperability is the ability of two or more systems and devices to cooperate and exchange data, and interpret that shared information2. An important goal is to make the best possible use of the existing and upcoming wealth of genetic, genomic, and phenotypic data in fundamental and applied wheat science. Hence, data interoperability has become a hot topic in this community, given the ever-growing data deluge coming from improvements in data generating technologies and large-scale computational methods for handling DNA and RNA sequencing, high throughput genotyping and phenotyping, high throughput imaging, and satellite monitoring. However, achieving data interoperability is difficult not only because of data and tool heterogeneity, i.e., the ‘technical debt’, but also because of social and scientific issues, such as lack of curation experts, lack of value chains for data generators, and lack of a first class digital citizen recognition for data managers, i.e. the ‘cultural debt’.\n\nTo help address these debts, the Wheat Data Interoperability Working Group (WDI-WG, https://www.rd-alliance.org/groups/wheat-data-interoperability-wg) was created as one of the Research Data Alliance working groups (https://www.rd-alliance.org/groups), under the umbrella of the WheatIS Expert Working Group (http://wheatis.org/), which is endorsed by the IWI to build an international information system for wheat genetic, genomic and phenotypic data. The Working Group included wheat scientists, as well as ontologists and data experts from different organizations and countries, and its mission was to provide a common framework for describing and representing data with respect to existing open data standards. From the outset, the objective of the WDI-WG was to deter communities from creating new standards, which would have made the already-complex landscape of existing data standards even more complex. The WDI-WG collected valuable information through two surveys of the wheat research community, comprising responses regarding existing data formats, practices, and the use of ontologies and controlled vocabularies. From these surveys, the WDI-WG then developed a set of specific recommendations, and worked to facilitate data interoperability through the harmonization of data formats, data models and vocabularies usage, thus aiming to address the main interoperability issues. The proposed recommendations have been endorsed by the WheatIS Expert Working Group and the Technical Advisory Board of the RDA (RDA-TAB).\n\nThis paper describes the results and the collaborative methodology used by the WDI-WG, which we believe will be of interest to formalize data interoperability in other crop research communities.\n\n\nDeveloping the recommendations\n\nFrom the preparation to the publication of the recommendations, the WDI-WG strongly based its work on the wheat research community. Similarly, the maintenance of the recommendations will be reliant on feedback of the community and the review of a steering group, which includes representatives of the adopters of the guidelines. The main steps of the methodology adopted by the WDI-WG are represented in Figure 1 and are described in more detail in the rest of this section.\n\nThe WDI-WG standpoint was to build on prior practices in use in the community, reusing existing standards as much as possible. Gaps, if they existed, could then be filled through the development of new standards. This principle led the working group to start with two surveys, interrogating the wheat research community through the IWI communication channels. The first, “Data standards in the wheat research community wheat data interoperability WG”, studied the usage of data standards in the wheat research community through a series of questions sent out to researchers and stakeholders in wheat science. The questions and answers are summarized in a report11. The results allowed the group to identify the most commonly used data formats and controlled vocabularies in the wheat research community. The second survey, “Towards a Comprehensive Overview of Ontologies and Vocabularies for Research on Wheat\", focusing on ontologies and vocabularies, allowed the WDI-WG to collect information about the visibility, interoperability, domain, and content of relevant ontologies and vocabularies. The questions and answers of this survey are also summarized in a report12.\n\nTwo meetings of the WDI-WG were organized in 2014 and 2015, as well as regular face-to-face and online meetings, to analyze the survey results in order to draw recommendations. Calls for participation were regularly posted on the websites of RDA and IWI and channeled by the stakeholders. During these working sessions, wheat research scientists, data and information managers, and semantic web experts discussed and collectively agreed on a set of recommendations to cover the widest set of requirements of the communities they supported. The criteria used to guide the recommendation process were the following: (i) reuse existing standards and reinforce existing good practices with regards to interoperability to preserve synergies that work well in the community and (ii) promote emerging standards and practices where gaps exist.\n\nFor the following data types of interest to the WDI-WG, the surveys confirmed the existence of adequate consensus regarding data exchange formats: DNA sequence and any associated variants, genome/transcriptome annotations, gene expression data, and physical maps. As such, the WDI-WG recommended the formats that were the most used and/or compliant with the most popular tools and/or already interoperable with other data formats. For example, the GFF3 file format (http://gmod.org/wiki/GFF3) is found to be widely used by the community to represent genome annotations. Moreover, a Genbank-to-GFF3 script converter is available (http://www.hpa-bioinformatics.org.uk/biosnippets/snippets/115), in addition to a GFF3 validator tool (http://genometools.org/cgi-bin/gff3validator.cgi). Thus, the WDI-WG recommended GFF3 for the representation of genome annotations.\n\nHowever, unlike the aforementioned data types, the wheat data standards survey did not show good consensus for phenotypes and germplasm in terms of data exchange formats and data description practices. For these data types, the WDI-WG collectively agreed to recommend emerging standards, such as (i) Minimum Information About Plant Phenotyping Experiment (MIAPPE)4 and its ISA-TAB implementation4; (ii) the Crop Ontology5, especially the Wheat Trait Ontology for phenotypes (http://agroportal.lirmm.fr/ontologies/CO_321); and (iii) the FAO-IPGRI Multi-Crop Passport Ontology (http://agroportal.lirmm.fr/ontologies/CO_020) for germplasm.\n\nPrior to their endorsement by RDA, the resulting recommendations have been reviewed by the WheatIS expert working group. As a deliverable of a RDA working group, the recommendations received feedback from the RDA community and validation from the RDA-TAB.\n\nThe WDI-WG also used many of the available channels in order to obtain feedback from the wheat research community. In particular, feedback was requested, and was obtained, from communications through the Wheat Initiative’s website, the Food and Agriculture Organization Agricultural Information Management Standards (AIMS) newsletter, and various national and institutional mailing lists.\n\nThe recommendations are published on the b2share repository13, and a website (http://datastandards.wheatis.org), which provides the option to submit comments and suggestions. Thus, recommendations can be updated as required by the wheat community, which is of significance since technologies and practices are constantly evolving. Hence, this kind of media allows keeping the guidelines relevant and useful for the wheat research community.\n\n\nDisseminating the recommendations\n\nThe first and main output of the WDI-WG is a set of recommendations for describing, representing and linking wheat data. These recommendations are available at http://datastandards.wheatis.org and cover the following data types: sequence variations, genome annotations, phenotypes, physical maps, germplasm, and gene expression. The navigation menu of the website includes four main items (Figure 2): “Guidelines”, “Ontologies and vocabularies”, “Use cases”, and “Getting involved”. The guidelines menu contains a section for each of the data types addressed by the WDI-WG. Each data type-specific page (Figure 3) contains the following sections: (i) a summary of the recommendations for the indicated data type; (ii) rationalized recommendations about data format standards; (iii) rationalized recommendations about metadata standards and ontologies; (iv) tools; (v) examples; and (vi) comments. The summary of the recommendations13 and the http://datastandards.wheatis.org website provide detailed information for each data type covered by the guidelines.\n\nIn addition to the data type-specific pages, a page dedicated to ontologies and vocabularies explains their benefits and current situation in the context of wheat research data. The use cases page describes examples of use cases with interoperability issues.\n\nIn the context of research data, the use of common vocabularies or ontologies plays a key role in managing, publishing, and reusing data6. Words may have different meanings to different people, and standard definitions for these words are key to avoiding miscommunication and enabling good collaboration. Standardized vocabularies and ontologies enhance the efficiency of interoperability and the effectiveness of data exchange, thus facilitating the reuse of data by others5,7. A need to offer a common unique repository of standard vocabularies and ontologies relevant for wheat was identified, and the AgroPortal (http://agroportal.lirmm.fr/)8, a starting project in 2015, was recognized as suitable solution.\n\nAgroPortal is a collaborative initiative to build a repository of vocabularies and ontologies for agronomy, and related domains (plant sciences, biodiversity, and nutrition). By reusing the National Center for Biomedical Ontologies (NCBO) BioPortal technology9, the portal features ontology hosting, search, versioning, visualization, comment, recommendation, and enables semantic annotation, as well as storing and exploiting ontology alignments, all within a semantic web compliant infrastructure. The AgroPortal specifically pays attention to respect the requirements of the agronomy community in terms of ontology formats (e.g., SKOS, trait dictionaries), or supported features (metadata, annotation). AgroPortal addresses the WDI-WG identified need, while offering a set of interesting features for the ontologies being hosted. Therefore, we have created and maintain an explicit group within AgroPortal and its corresponding slice (http://wheat.agroportal.lirmm.fr/). Slices are a mechanism supported by the platform to allow users to interact (both via user and application programming interfaces) only with a subset of ontologies in AgroPortal. If browsing the slice, all the portal features will be restricted to a subset, enabling users to focus on their specific use cases. As of today, AgroPortal’s wheat group contains 20 ontologies of the 23 identified by the WDI-WG10. Each ontology has been carefully described (with licenses, authority, availability, etc.), and a new metadata property (omv:endorsedBy) is used to show the ontology’s endorsement by the WDI-WG. The wheat slice in AgroPortal will allow the community to share common meanings of the words they utilize to describe and annotate data, which will in turn make the data more machine-readable and interoperable. Furthermore, the slice will enable wheat-related ontology developers to make their ontologies more visible to the agronomic research community, thus contributing to reduce the proliferation of concurrent ontologies on the Web. This slice has been reported in the WDI-WG guidelines web site (section “Ontologies and vocabularies”), and used as a reference resource to identify and select ontologies related to wheat since then.\n\n\nDiscussion\n\nThe WDI-WG’s guidelines have been collaboratively built and validated under the umbrella of two authoritative organizations (the WheatIS expert working group and RDA, respectively). The Expert Working Groups of the Wheat Initiative have been instrumental in efficiently interacting with the wheat scientific community in order to take into account the needs from the different fields of biology working on this species. Consequently, the needs and the practices of this community were well-addressed. In addition, the WDI-WG took care to build on existing good practices and preserve prevailing strong synergies in the community, while proposing new standards and practices where relevant. This has been achieved by consulting the wheat research community and experts as frequently as needed. This strategic approach ensures a better adoption of the guidelines by the wheat research community.\n\nDespite this initial strong validation process, the WDI-WG anticipates changes to the recommendations, especially due to an evolving landscape of standards and practices. The blog-like website that hosts the recommendations will facilitate rapid implementation of future changes.\n\nOne pitfall the WDI-WG managed to avoid is the quest for immediate comprehensiveness. We deliberated focused on the six data types that were considered most relevant by the wheat research community in the coming years. However, the recommendations can be extended to more data types in future.\n\nIn order to maximize the adoption of the recommendations, the WDI-WG favors a bottom-up approach rather than enforcing the choice of particular standards. Consequently, to begin with, it is better that individual project initiatives develop their own usage of the proposed recommendations and standards, especially since there are some standards that share common concepts, but address different needs. We prefer the community to adopt at least some standards rather than none. We provide guidelines to facilitate the decision of standards suggesting the most widely adopted ones. By developing the tools required to map/convert from one standard to another, it should be possible to bridge data respecting different standards. The important point is that a standard is used to remove ambiguities in data semantics and representation to enable automated processing. At a later date, when several standards have converged or become widely adopted, it could be possible to enforce their usage. But the time needed to reach this second step will vary between the different fields of biology.\n\nThe WDI-WG will develop training programs to increase the adoption of the guidelines. In fact, the guidelines have already been adopted by a number of stakeholders (http://ist.blogs.inra.fr/wdi/adopters/). However, these are part of large institutions. This highlights the need to provide tools and training to facilitate the adoption of the guidelines within smaller organizations. Two kinds of training will be developed for two types of audience: data managers with technical skills on data management, and biologists with data knowledge. Another target community of adopters of the WDI-WG’s guidelines is software developers. The adoption of the guidelines by this community is essential to showcase the benefits of data interoperability. Therefore, there is a strong need to raise awareness in this community.\n\nFinally, reengineering legacy data in accordance with the WDI-WG is an open question. Indeed, it requires from data producers and managers to convert legacy data in recommended data formats or learn how to annotate data with specific vocabularies, which is not trivial for anyone. Depending on the use case and/or the value of the data, it may or may not be worth making such efforts. The use of automated tools for the transformation of data in different formats (where applicable) is expected to minimize the human effort required for such processes.\n\n\nFollow up and conclusions\n\nAs an RDA working group, the WDI-WG is now in an adoption and maintenance phase. Consequently, the WDI-WG will know focus on dissemination and maintenance activities. A steering group, including representatives of the adopters and the WDI-WG chairs, will drive these activities, taking into account the feedback and contributions of the wheat research community. The action plan of the WDI-WG includes: (i) the promotion of the guidelines via development of information material such as flyers or short videos; and (ii) technical and non-technical training for data managers and scientists, respectively. The WDI-WG will also consolidate the wheat vocabularies group and slice within AgroPortal (http://wheat.agroportal.lirmm.fr/ontologies). In addition to these projects, it is worth mentioning that the methodology and the results of the WDI-WG have inspired the creation of a rice data interoperability working group within the frame of RDA (https://www.rd-alliance.org/groups/rice-data-interoperability-wg.html).\n\nThe recommendations of the WDI-WG are intended for data producers, data managers, data consumers, and software developers. They constitute a key building block for FAIR data1 sharing infrastructures (https://www.force11.org/fairprinciples). Indeed, the adoption of the recommendations will facilitate the depositing of data within well recognized repositories in addition to make them easily understandable and reusable.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe WDI-WG was partially funded by IWI funding received by the WheatIS Expert Working Group.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nReferences\n\nWilkinson MD, Dumontier M, Aalbersberg IJ, et al.: The FAIR Guiding Principles for scientific data management and stewardship. Sci Data. 2016; 3: 160018. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWegner P: Interoperability. ACM Comput Surv. 1996; 28(1): 285–287. Publisher Full Text\n\nAubin S, Alaux M, Baumann U, et al.: Data standards in the wheat research community. Zenodo. 2014. Publisher Full Text\n\nĆwiek-Kupczyńska H, Altmann T, Arend D, et al.: Measures for interoperability of phenotypic data: minimum information requirements and formatting. Plant Methods. 2016; 12: 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShrestha R, Arnaud E, Mauleon R, et al.: Multifunctional crop trait ontology for breeders’ data: field book, annotation, data discovery and semantic enrichment of the literature. AoB Plants. 2010; 2010: plq008. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRubin DL, Shah NH, Noy NF: Biomedical ontologies: a functional perspective. Brief Bioinform. 2008; 9(1): 75–90. PubMed Abstract \| Publisher Full Text\n\nJaiswal P, Cooper L, Elser JL, et al.: Planteome: A resource for Common Reference Ontologies and Applications for Plant Biology. In Plant and Animal Genome XXIV Conference. Plant and Animal Genome. 2016. Reference Source\n\nJonquet C, Toulet A, Arnaud E, et al.: Reusing the NCBO BioPortal technology for agronomy to build AgroPortal. 7th Int Conf Biomed Ontol. 2016. Reference Source\n\nNoy NF, Shah NH, Whetzel PL, et al.: BioPortal: ontologies and integrated data resources at the click of a mouse. Nucleic Acids Res. 2009; 37(Web Server issue): W170–W173. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubirats I, Cooper L, Shrestha R, et al.: Towards a Comprehensive Overview of Ontologies and Vocabularies for Research on Wheat. Zenodo. 2015. Publisher Full Text\n\nAubin S, Alaux M, Baumann U, et al.: Data standards in the wheat research community. Zenodo. 2014. Publisher Full Text\n\nSubirats I, Cooper L, Shrestha R, et al.: Towards a Comprehensive Overview of Ontologies and Vocabularies for Research on Wheat. Zenodo. 2015. Publisher Full Text\n\nDzalé Yeumo E, Fulss R, Alaux M, et al.: Wheat Data Interoperability Guidelines, Ontologies and User Cases. Recommendations from the RDA Wheat Data Interoperability Working Group. EUDAT B2Share. Publisher Full Text" }	[ { "id": "27011", "date": "30 Oct 2017", "name": "Ramil Mauleon", "expertise": [ "Reviewer Expertise Bioinformatics", "data standards", "genetics", "genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe opinion article is well written, and addresses a challenge that plant/crop science researchers face: how to systematically manage data from high throughput technologies (ie. next-gen sequencing, high density genotyping, standardized phenotype measurements/observations)\nThe inventory made of data standards dealing with wheat research (with greater focus on genomics/sequences and lesser on phenotype and germplasm, as acknowledged by authors) is very comprehensive and I believe covers what most of the wheat community is using.\nSome minor improvements I see that could be done on the main paper itself is to mention directly some important summaries /findings of the survey results, without having to open the links to the results of the survey. For example, a reader might wish to know how many (or what proportion) of the wheat research institutions surveyed have data standards of what kind (making the paper citable directly and showing the importance of the WDI-WG paper). This is one of the few summaries that could be directly shown. Another is the mention of data standards for genotyping data (especially high density ones), directly in the paper, this is very important data type , I had to open the survey to know more about this.\nReaders who wish to use the recommendations would also likely benefit from having concrete examples of documents that implement the recommendations of the paper directly available (again without having to navigate the external website of the cited resources) within the paper itself. Example, a direct example of snippet of GFF3 for a particular genome annotation would be nice. Can you also mention the most recent resource for wheat genome build, the most authoritative (or most widely used) gene naming of wheat genome (as of writing)? This is very important info this type of data. Another example could be a snippet of a phenotyping experiment result (a field book table, for example), wherein the MIAPPE terms, and the ontology terms tagging the phenotyping data observed/measured appropriate for the experiment can be seen? This gives the reader/researcher ideas on how the data standards/guidelines are used in real-world applications. I went to the http://datastandards.wheatis.org/ externally referred site and I did not easily see a sample dataset that could be used as template.\n\nIn summary, the paper is already in a very mature and good state, just having some important summaries of the survey directly mentioned and providing examples of applications of the standards in easily accessible sample documents would be a welcome addition.\nBest wishes to the authors!\n\nIs the topic of the opinion article discussed accurately in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nAre arguments sufficiently supported by evidence from the published literature? Yes\n\nAre the conclusions drawn balanced and justified on the basis of the presented arguments? Yes", "responses": [ { "c_id": "3203", "date": "06 Dec 2017", "name": "Hadi Quesneville", "role": "Author Response", "response": "Dear Ramil, Thank you for this positive review and your useful comments. We have modified the manuscript taking into account your suggestions as follows. We added a summary of the survey results as supplementary material to facilitate the reading of our article. In particular to provide the user with the current usage of the standard by the wheat community. Concerning examples of documents that implement the recommendations, we consider that this is out of the scope of our paper which describes the methodology we followed to propose standards and guidelines, and not what they are. Because of the nature of the recommendation that would evolve with time, we preferred to implement a website that could be updated according to the community usage as explained in the paper. Writing them in a paper would freeze them and this is contrary to our philosophy. The examples can be found on our guideline website. Best regards," } ] } ]	1	https://f1000research.com/articles/6-1843
https://f1000research.com/articles/4-1/v3	15 May 17	{ "type": "Method Article", "title": "Quantification of polysaccharides fixed to Gram stained slides using lactophenol cotton blue and digital image processing", "authors": [ "Bryan Ericksen" ], "abstract": "Dark blue rings and circles emerged when the non-specific polysaccharide stain lactophenol cotton blue was added to Gram stained slides. The dark blue staining is attributable to the presence of capsular polysaccharides and bacterial slime associated with clumps of Gram-negative bacteria. Since all bacterial cells are glycosylated and concentrate polysaccharides from the media, the majority of cells stain light blue. The contrast between dark and light staining is sufficient to enable a digital image processing thresholding technique to be quantitative with little background noise. Prior to the addition of lactophenol cotton blue, the Gram-stained slides appeared unremarkable, lacking ubiquitous clumps or stained polysaccharides. Adding lactophenol cotton blue to Gram stained slides is a quick and inexpensive way to screen cell cultures for bacterial slime, clumps and biofilms that are invisible using the Gram stain alone. The presence of cell clumping provides a possible explanation of the presence of persisters and paradoxical points observed in Virtual Colony Count antimicrobial assays, and suggests a phenotypic resistance mechanism to antimicrobial peptides involving capsular polysaccharides.", "keywords": [ "Antimicrobials", "Biofilms", "E. coli", "Environmental" ], "content": "Introduction\n\nThe virtual colony count (VCC) microbiological assay has been used for over a decade to measure the effect of antimicrobial peptides such as defensins and LL-37 against a variety of bacteria. (Ericksen et al., 2005; Zhao et al., 2013). It infers antimicrobial activity based on the quantitative growth kinetics of 200 μL batch cultures of bacteria grown in 96-well plates using a method of enumeration of viable cells (Brewster, 2003) mathematically identical to the method of enumeration of amplicons utilized by quantitative real-time PCR (Heid et al., 1996). The originally published plate configuration included a ring of 36 wells containing uninoculated Mueller Hinton Broth (MHB) capable of detecting cross-contamination (Ericksen, 2014b). There was evidence that bacteria might form clumps and biofilms during the assay, including scatter detectable by the plate reader and the presence of ubiquitous macroscopic clumping in tryptic soy broth. 10 μL samples of cross-contamination control wells that had become turbid after VCC experiments were Gram-stained, revealing few clumps. Apparently, most clumps were not retained on the glass during the Gram stain procedure (Gram, 1884), whether fixed to the slide by heat or methanol. The application of lactophenol cotton blue, ordinarily used to visualize fungi by staining cell wall polysaccharides such as chitin, revealed circles and rings consistent with the caramelized residue of polysaccharides, which presumably included capsular polysaccharides and slime secreted concomitantly with clump and biofilm formation. These dark blue circles and rings could be consistent either with a heterogeneous subpopulation of E. coli or with slight contamination with a second strain.\n\n\nMaterials and methods\n\nThe VCC assay was conducted using the 36 edge wells to detect contamination as originally described (Ericksen et al., 2005), except a rectangular piece of Parafilm M (6 × 0.25 squares) was wrapped around the 96-well plate before the start of the 2-hour and 12-hour plate reader runs. Parafilm strips remained almost entirely intact and in place throughout the 12-hour run at 37°C and resulted in the complete absence of dust large enough to be visible using an Olympus 8Z61 crystallographic microscope on the ledge between the 96 wells and the edge of the plate, except for a single speck in one experiment observed near a crack in the Parafilm. Parafilm also prevented the visible decrease in edge well volume due to evaporation that originally necessitated excluding these wells from the experimental portion of the assay (Ericksen et al., 2005). This evaporation also caused a slight progressive increase in optical density to a maximum ΔOD650 of 0.004 among the edge wells over the course of the 12-hour experiment as the Mueller Hinton Broth became more concentrated. This evaporation was too slight to affect experimental (inoculated) wells measurably or affect the linearity of the calibration curve. 10 µL samples of edge wells were added to droplets of sterile water or media and spread on Mueller Hinton Agar, Tryptic Soy Agar, and Sabouraud’s Agar plates. Colonies were analyzed by morphology, wet mounts, Gram stains, and biochemical analysis using Becton Dickinson Enteropluri Product Number 261185.\n\nLactophenol Cotton Blue Gram Stain Procedure. Overnight steps allowed for equilibration to the ambient humidity during summer months in the IHV building at UMB, which ranged from 40–60%. Water content and temperature may be important factors for the caramelization process to be quantitatively reproducible.\n\nGlass slides were scrubbed with PCMX hand soap using a pipe cleaner. 10 µL of cells sampled from 96-well plates after VCC assays using twice-concentrated MHB in the outgrowth step were added to the slides and equilibrated to ambient humidity overnight. The slides were heat-fixed by placing the sample at the point in space at the upper tip of the inner blue flame of a Bunsen burner three times for one second each, removing the slide for one second in between (Figure 1). Ambient relative humidity was 40–60%. The slides were stained with Fluka Analytical Gram Staining Kit Product Number 77730 and again equilibrated to ambient humidity overnight in a vertical position. Becton Dickinson Lactophenol Cotton Blue Stain Droppers Product Number 261188 were applied to the Gram stained sample and digital images were captured using an Amscope light microscope at 160×, 400× and 1600× magnification and Toupview software. The Adobe Photoshop thresholding function was applied to the 400× digital images using a threshold of 100. Black pixels were enumerated using the histogram function.\n\n\nResults\n\nMacroscopic clumps were observed in 25 mL TSB batch cultures of E. coli ATCC® 25922™ grown at 37°C in early exponential phase to an expected optical density at 650 nm (OD650) of approximately 0.3. A 1 mL uncovered sample placed in a cuvette and cooled to room temperature rapidly formed small macroscopic clumps (up to about 1 mm in diameter), some of which exhibited motility, swimming in a synchronized wave downward to form a single large macroscopic clump (up to 1 cm long, equal to the cuvette width) at the base of the cuvette. OD650 plummeted up to 2% per minute, reaching equilibrium after a 10–20% decrease when placed in a room temperature HPLC detector, as cells in suspension joined the clump beneath the light path. The optical density readings declined so rapidly that only the first two digits of the four reported by the Waters 600 detector could be recorded. Observing cuvettes containing such clumps, it was apparent that cohesion, rather than adhesion, was more important, since the clumps moved downward from one corner to the other corner of the cuvette as it was rotated by hand.\n\nMacroscopic clumping in the batch culture or cuvette outside the detector was no longer observed after four changes: 1. using a small HEPA-filtered air purifier, 2. replacing in-house deionized Milli-Q water with purchased molecular biology grade water, 3. replacing 2×MHB prepared and autoclaved in-house using reusable jars with Teknova 2× cation-adjusted MHB, and 4. filter-sterilizing phosphate buffers made near the portable air purifier, rather than autoclaving in reusable jars. Even after these remediation measures, uncovered 1 mL samples placed in the detector for 2 hours formed a macroscopic clump at the base of the cuvette accompanied by a decrease in optical density, suggesting that at least one clumping environmental factor (CEF) was concentrated by the fan and filter within detector acting as a dust trap. Thus, 1 mL samples of E. coli ATCC® 25922™ served as biosensors for CEFs, and the detector served as a biosensor positive control.\n\nCorner-seeking motility of E. coli ATCC® 25922™ was also observed on MH agar plates wrapped in Parafilm and incubated at room temperature for 2–3 weeks, as indicated by the formation of a ~1 cm-wide confluent ring around the entire edge of the plate, even though confluent areas and single colonies that originally appeared after 1–2 days were separate from the edge.\n\nThe UMB VCC procedure was sensitive to cross-contamination in the 36 uninoculated edge wells, possibly indicating that clumping affects the particle size distribution and adhesive properties of the cells, which in turn promotes aerosol formation during pipetting (Ericksen, 2014b). Figure 2 depicts cells sampled from a cross-contaminated edge well after storage at 4°C. The UCLA VCC method, with cells in 10 µL pipetted beneath 90 µL rather than a 50 µL suspension added to 50 µL as droplets from above, (Welkos et al., 2011) minimizes the probability of cross-contamination and is a safer and more effective method of transferring bacteria such as the hazardous BSL-3 pathogen Bacillus anthracis.\n\nA: Blue rings indicate the polysaccharide residue of clumps of cells presumably washed from the slides during the Gram stain procedure. These polysaccharides were invisible when inspected after Gram staining and before application of lactophenol cotton blue. Other experiments produced smaller dark blue circles rather than rings. B: Thresholding results. A large majority of black pixels are contained within the polysaccharide rings.\n\nThe lactophenol cotton blue Gram stain (BGS) revealed ubiquitous circular or ring-shaped structures that stained dark blue (Figure 2A). All cells stained light blue because all cells are glycosylated and concentrate polysaccharides from the media as part of their metabolism. Rare regions of indistinct blue staining were also observed, probably resulting from starch and other polysaccharides present in MHB, suggesting that the intensity of blue staining could also arise from starch and other carbohydrates with the capsular polysaccharides. MHB contains 1.5 g/L starch, plus a variety of other carbohydrates contained in beef extract. Carbohydrates, which must have included Maillard reaction (Maillard, 1912) and caramelization products, adhered to the glass in the intense heat of the fixation steps and endured on the slide throughout the Gram stain procedure. These polysaccharide residues had been invisible when these same slides were observed after Gram staining and before application of lactophenol cotton blue. The intensity of dark blue staining suggests copious capsule and slime formation.\n\nApplying the thresholding technique using a threshold of 100 differentiated the dark from the light staining with little apparent background noise (Figure 2B). Thresholding of BGS images captured at 160× and 1600× magnification (Figure 3) are also possible using the Amscope microscope. However, pixelation could add imprecision at 160× and the large size of clumps would increase variability from field to field at 1600×. TSB or MHB cultures of E. coli ATCC® 43827™ (ML-35) produced no macroscopic clumps under any conditions in several experiments conducted in 2013 and 2014, indicating that the observed clumping is strain-dependent.\n\nA–C: 160×. D–F: 400×. G–I: 1600×. Cells were sampled from the edge wells of a different virtual colony count experiment than Figure 2.\n\nThe history of hundreds of VCC experiments at UMB between 2003 and 2014 (Ericksen et al., 2005; Pazgier et al., 2012; Rajabi et al., 2012; Wei et al., 2009; Wei et al., 2010; Wu et al., 2005; Wu et al., 2007; Xie et al., 2005a; Xie et al., 2005b; Zhao et al., 2012; Zhao et al., 2013; Zou et al., 2008) clearly shows that edge wells are almost always clear, not turbid, after the 12h outgrowth phase of VCC experiments. In a 1-month period in August and September 2013, 13 quadruplicate calibration experiments were conducted using the same pipetting technique as the sextuplicate calibration experiments in the original VCC publication (Ericksen et al., 2005). However, in the 2013 experiments, four, rather than six, calibration curves were confined to 32 internal wells (C3-F10). These experiments used the rich media MHB, TSB or slight variations thereof. The external 64 wells (rows A, B, G and H and columns 1, 2, 11 and 12) contained two rings of contamination control wells rather than the single ring of 36 wells originally used. In these experiments conducted just outside a biosafety cabinet used for VCC experiments, none of the 832 contamination control wells turned turbid after the 12h incubation. Assuming clumping is caused by an environmental factor, these experiments strongly suggest that CEFs present in the laboratory environment are overwhelmingly non-culturable in rich media such as MHB or TSB. An alternate explanation of infrequent cell clumping and rare paradoxical points is that bacterial cells have a mechanism to induce clumping and biofilm formation infrequently and constitutively even in the absence of any causative agent or contaminant. If cell clumping is caused by a contaminant, several possible sources are present in the laboratory environment. In addition to viable contamination, unculturable bacteria could exert an influence upon rapidly growing E. coli cells. Furthermore, nucleic acids are known to cause cells to coalesce into clumps over a broad size distribution in both bacterial and mammalian cell culture. Airborne CEFs smaller than a bacterial cell could pass through the HEPA filters with little or no resistance, meaning that these molecules could have affected experiments conducted both inside and outside biosafety cabinets. Measures such as trypsinization, treatment with other proteases, and treatment with nucleases such as benzonase are commonly employed to reduce or eliminate clumping (Kruse & Patterson, 1973). For the same purpose, shear was employed in VCC calibration curves by placing pipette tips in contact with the cross-sectional corner of each well when pipetting up and down 15 times to mix (Ericksen, 2014b), although growth curves showed evidence of clumps large enough to produce measurable differences in optical density that preceded exponential growth. Clumping had no effect on the linearity of the calibration curve, possibly indicating that a small fraction of cells routinely grow as clumps and biofilms in the absence of antimicrobial agents.\n\n\nDiscussion\n\nThe presence of polysaccharides associated with E. coli ATCC® 25922™ cohesion suggests that in the conditions studied at UMB, this strain employs clumping, possibly as a defense mechanism. Forming a clump surrounded by polysaccharides could contribute to resistance to antimicrobial lectins such as defensins (Wang et al., 2003) that would be bound and inhibited at the surface, limiting further inward diffusion and protecting persister cells (Ericksen et al., 2005) at the center of the clump. These survivors could contribute to the deviation from simple exponential killing (Luria & Latarjet, 1947) observed throughout all VCC studies at UMB of defensin activity against E. coli. They could also explain the presence of paradoxical data points observed occasionally throughout the history of VCC experiments at UMB. For example, the defensin HNP1 at the highest concentration of 256 µg/mL caused greater survival than 128 µg/mL in the initial VCC study (Ericksen et al., 2005) MHB contains a considerable amount (1.5 g/L) of added starch. Polysaccharides in rich media could contribute to the complete inhibition of antimicrobial peptides, which is essential for VCC assays to be capable of enumerating surviving bacteria by the QGK data analysis method. Qualitative defensin lectin activity generally follows the hierarchy: glycosylated proteins > branched polysaccharides > linear polysaccharides > oligosaccharides > monosaccharides. (Lehrer, R. I., personal communication) Bacterial slime and capsules are highly branched and contain glycosylated proteins (Wilkinson, 1958). If inhibition follows the same qualitative pattern as binding, bacterial capsular polysaccharides would be potent defensin inhibitors. Clump, biofilm and capsule formation may have evolved partially as resistance mechanisms to the ancient selection pressure exerted throughout the tree of life by antimicrobial peptides in the environment.\n\nA possible consequence of the inhibition of defensins by polysaccharides could be that therapies with lectin antimicrobial peptides as active ingredients would not be effective against clumps or biofilms in the absence of at least one other active ingredient that degrades the polysaccharide capsule, such as a glycosidase. Because polysaccharide structures in capsules and slime vary widely, as do glycosidase substrate specificities, any given enzyme might be active against only a narrow range of bacteria. In the absence of in vivo glycosidases, activity against a broad spectrum of pathogenic bacteria would therefore require an enzyme cocktail of glycosidases accompanying the lectin antimicrobial peptide or a glycosidase with unusually promiscuous substrate specificity.\n\n\nData availability\n\nfigshare: Blue Gram Stain images from three cross-contamination edge wells of a Virtual Colony Count assay at 160×, 400×, or 1600×, doi: http://dx.doi.org/10.6084/m9.figshare.1269193 (Ericksen, 2014a).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author acknowledges Peprotech, Inc. for funding this work.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nI thank Corinne Delaney Tochman for the graphic design of Figure 1, and Robert I. Lehrer for helpful comments.\n\n\nReferences\n\nBrewster JD: A simple micro-growth assay for enumerating bacteria. J Microbiol Methods. 2003; 53(1): 77–86. PubMed Abstract \| Publisher Full Text\n\nEricksen B: Blue Gram Stain images from three cross-contamination edge wells of a Virtual Colony Count assay at 160×, 400×, or 1600×. figshare. 2014a. Data Source\n\nEricksen B: Safety, efficacy and utility of methods of transferring adhesive and cohesive Escherichia coli cells to microplates to avoid aerosols [v2; ref status: indexed, http://f1000r.es/4yt]. F1000Res. 2014b; 3: 267. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEricksen B, Wu Z, Lu W, et al.: Antibacterial activity and specificity of the six human {alpha}-defensins. Antimicrob Agents Chemother. 2005; 49(1): 269–275. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGram C: The Differential Staining of Schizomycetes in Tissue Sections and in Dried Preparations. Fortschitte der Medicin. 1884; 2: 185–189. Reference Source\n\nHeid CA, Stevens J, Livak KJ, et al.: Real time quantitative PCR. Genome Res. 1996; 6(10): 986–994. PubMed Abstract \| Publisher Full Text\n\nKruse PF, Patterson MK: Tissue culture: methods and applications. Academic Press, New York. 1973. Reference Source\n\nLuria SE, Latarjet R: Ultraviolet Irradiation of Bacteriophage During Intracellular Growth. J Bacteriol. 1947; 53(2): 149–163. PubMed Abstract \| Free Full Text\n\nMaillard LC: Action of Amino Acids on Sugars. Formation of Melanoidins in a Methodical Way. Compt Rend. 1912; 154: 66. Reference Source\n\nPazgier M, Wei G, Ericksen B, et al.: Sometimes it takes two to tango: contributions of dimerization to functions of human α-defensin HNP1 peptide. J Biol Chem. 2012; 287(12): 8944–53. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRajabi M, Ericksen B, Wu X, et al.: Functional determinants of human enteric α-defensin HD5: crucial role for hydrophobicity at dimer interface. J Biol Chem. 2012; 287(26): 21615–27. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWang W, Cole AM, Hong T, et al.: Retrocyclin, an antiretroviral theta-defensin, is a lectin. J Immunol. 2003; 170(9): 4708–4716. PubMed Abstract \| Publisher Full Text\n\nWei G, de Leeuw E, Pazgier M, et al.: Through the looking glass, mechanistic insights from enantiomeric human defensins. J Biol Chem. 2009; 248(42): 29180–92. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWei G, Pazgier M, de Leeuw E, et al.: Trp-26 imparts functional versatility to human alpha-defensin HNP1. J Biol Chem. 2010; 285(21): 16275–85. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWelkos S, Cote CK, Hahn U, et al.: Humanized theta-defensins (retrocyclins) enhance macrophage performance and protect mice from experimental anthrax infections. Antimicrob Agents Chemother. 2011; 55(9): 4238–4250. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWilkinson JF: The extracellualr polysaccharides of bacteria. Bacteriol Rev. 1958; 22(1): 46–73. PubMed Abstract \| Free Full Text\n\nWu Z, Li X, de Leeuw E, et al.: Why is the Arg5-Glu13 salt bridge conserved in mammalian alpha-defensins? J Biol Chem. 2005; 280(52): 43039–47. PubMed Abstract \| Publisher Full Text\n\nWu Z, Li X, Ericksen B, et al.: Impact of pro segments on the folding and function of human neutrophil alpha-defensins. J Mol Biol. 2007; 368(2): 537–49. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nXie C, Prahl A, Ericksen B, et al.: Reconstruction of the conserved beta-bulge in mammalian defensins using D-amino acids. J Biol Chem. 2005a; 280(38): 32921–9. PubMed Abstract \| Publisher Full Text\n\nXie C, Zeng P, Ericksen B, et al.: Effects of the terminal charges in human neutrophil alpha-defensin 2 on its bactericidal and membrane activity. Peptides. 2005b; 26(12): 2377–83. PubMed Abstract \| Publisher Full Text\n\nZhao L, Ericksen B, Wu X, et al.: Invariant gly residue is important for α-defensin folding, dimerization, and function: a case study of the human neutrophil α-defensin HNP1. J Biol Chem. 2012; 287(23): 18900–12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZhao L, Tolbert WD, Ericksen B, et al.: Single, double and quadruple alanine substitutions at oligomeric interfaces identify hydrophobicity as the key determinant of human neutrophil alpha defensin HNP1 function. PLoS One. 2013; 8(11): e78937. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZou G, de Leeuw E, Lubkowski J, et al.: Molecular determinants for the interaction of human neutrophil alpha defensin 1 with its propeptide. J Mol Biol. 2008; 381(5): 1281–91. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "22739", "date": "14 Jun 2017", "name": "Klaus Kayser", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a good article that describes a new method to demonstrate E. coli cultures. The physical (laboratory) technique is well described and can easily reproduced. The virtual images, segmentation methods and colony identifications are good in principle, however, they could be explained more in detail. Especially detailed discussions of Regions of Interest (ROIs), segmentation algorithms, and potential expansion to improved interpretation could remarkably improve the reader's interest. Here are some articles mentioned that describe and discuss these aims and algorithms:\nKayser, K., B. Molnar, and R. Weinstein, 20061\nKayser, K., et al., 2016 2\nSharma, H. et al., 2015 3\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "2795", "date": "16 Jun 2017", "name": "Bryan Ericksen", "role": "Author Response", "response": "Thank you for your references to several interesting articles in the field of histopathology that describe sophisticated algorithms for image segmentation. The Blue Gram Stain, however, is not a histologic method, and it is important to make the distinction between a microbiological culture where objects are allowed to float freely relative to one another in solution and a histologic slide that is the result of paraffin embedding and thin sectioning, where geometry is much more relevant. It is also important to make a distinction between a region that stains dark blue as the result of the Blue Gram Stain, which presumably indicates polysaccharides such as bacterial slime, and cellular structures such as nuclei that stain darkly in histologic stains such as hematoxylin and eosin. Thresholding is the simplest form of segmentation. The more complex algorithms referenced in The Diagnostic Pathology Journal articles would not be applicable to the Blue Gram Stain, in which dark staining highlights relatively amorphous chemical residues, not spatially organized biological structures. Thank you also for suggesting the addition of a detailed discussion of regions of interest. I will do so here, referring to the figshare image names. Many of the slides depict similar fields, with rings of dark blue staining indicating polysaccharide residues fixed to the slides. A clear example of such an image is 400x-8.bmp, which shows blue rings of varying sizes, which in all cases are substantially larger than a single cell. The ring shape could indicate that a clump of bacteria had been present at that position, surrounded by a slime layer. During the subsequent steps of the Gram stain procedure, each clump was washed from the slide, carrying capsular polysaccharides in the center of the clump with it and leaving only a ring-shaped residue of slime behind on the slide. Several artifacts of the procedure are also apparent from these images. A large dark blue object is present in the lower right quadrant of image 400x-5.bmp, and a much smaller such region is apparent in the lower right quadrant of image 160x-3.bmp. These objects are the result of contamination that results from the manufacture of the glass slides used for this study, which necessitated scrubbing the slides with soap and a pipe cleaner before use. This contaminant was present in slides purchased from all five different manufacturers tested, even though the slides were marketed as “prewashed”. Scrubbing greatly reduced the frequency of this type of contamination. On rare occasion, staining appeared somewhat purple rather than blue, such as in image 160x-1.2.bmp, which presumably was the result of color distortion introduced by the microscope frame capture hardware. It is noted that the background of the slides is light blue, not white, indicating some very light staining due to the starch and other polysaccharides present in Mueller Hinton Broth; starch may also cause intermediate blue staining that does not appear to correspond to cell clumping, such as in images 160x-2 and 160x-3. Finally, black circles in images 160x-1.bmp, 160x-2.bmp, 160x-3.2.bmp and 160x-4.bmp are the result of air bubbles trapped beneath the coverslip. These can be avoided by omitting the coverslip, and must be absent from images used for quantitative digital image processing by thresholding. Finally, when asked whether all source data was available underlying the results to ensure full reproducibility, you responded “partly”. I assure the reader that the figshare contains a comprehensive set of images. All representative images were included, even those showing experimental artifacts, and the figshare includes a fairly large set of images (51). Also, as you mentioned, the laboratory technique can be easily reproduced from the description in this article. Therefore, this set of source data should be regarded as complete." } ] }, { "id": "23949", "date": "10 Jul 2017", "name": "Venkataramana Kandi", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis technical report is very interesting. Although the author used a simple experiment to demonstrate slime production, further researches need to done to evaluate such studies. In a clinical microbiology laboratory, this technique might well be of great use to preliminary identify, if a microorganism has the potential to form slime or bio-film. This may help us to analyze the reason, if any, in case the patient is not responding to the antibiotic (s) currently being used\nDrawback of the study includes, uncertainties about exactly what causes such observations under this special staining modification. Also it is unknown if the smear preparation has to be taken from broth or from the colonies. In case of colonies, what should be exact method to pick, since while routine smear preparation might interfere in the demonstrability of the slime, and you can almost never see biofilm,\nThis character is demonstrated by bacteria only when there is a demand, example, while the bacteria is inside human, fighting immune system to establish and cause infection.\nI appreciate the attempt of the author, though!\n\nIs the rationale for developing the new method (or application) clearly explained? Yes\n\nIs the description of the method technically sound? Yes\n\nAre sufficient details provided to allow replication of the method development and its use by others? Yes\n\nIf any results are presented, are all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions about the method and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3224", "date": "06 Dec 2017", "name": "Bryan Ericksen", "role": "Author Response", "response": "Thank you for approving this brief Method Article and for your thoughtful comments. You mentioned several drawbacks of the study. I hope the revisions in Version 5 have addressed each of them. Regarding uncertainty about what causes these observations under this special staining modification, although it seems a safe assumption that polysaccharides are being stained, it is unclear why the staining is ring-shaped. In the caption to Figure 2, I mentioned that staining produced “smaller dark blue circles” in other experiments; I have added the detail “filled-in circles” to Version 5. I also added to the “Regions of interest” section, “It is unclear why staining appeared as rings in some experiments and filled-in circles in other experiments. Perhaps subtle variations in the heat fixation or Gram stain steps resulted in clumps carrying polysaccharides from the center of each ring with them as they are washed away in some cases but not others.” This is a question that should be addressed further should these same two staining morphologies be discovered by other researchers who attempt to repeat these results. Regarding whether smear preparations need to be taken from broth cultures or colonies, I only tested broth cultures, since my purpose was to investigate cross-contamination in VCC assays, in which cells are grown in 200 microliter batch cultures on 96-well plates. I emphasized this detail in Version 4, and it is still reflected in Version 5. You mentioned that bacteria typically secrete slime only when there is a demand for such slime, such as in the human host fighting the immune system. I agree that slime formation may help bacteria evade the innate immune system, and it would be tempting to suggest that one of the ways that they do so is by secreting capsular polysaccharides to bind and inhibit lectin antimicrobial peptides such as the defensin HNP1. However, since I have not demonstrated this effect in the laboratory I would prefer not to mention it in the main text of this brief Method Article, which has already been criticized by another referee as being too speculative. I did, however, cite Robert I Lehrer’s studies of the lectin activities of defensins as a personal communication in the discussion. It would be helpful to emphasize that the cells in VCC cross-contamination control wells were not exposed to antimicrobial agents, and therefore the ubiquitous nature of the polysaccharide rings is somewhat surprising. I have added a sentence to this effect in the “Regions of interest” section of the results, and also mentioned that cells stained light blue appear to be planktonic." } ] } ]	3	https://f1000research.com/articles/4-1
https://f1000research.com/articles/5-2588/v1	27 Oct 16	{ "type": "Software Tool Article", "title": "exprso: an R-package for the rapid implementation of machine learning algorithms", "authors": [ "Thomas Quinn", "Daniel Tylee", "Stephen Glatt", "Daniel Tylee", "Stephen Glatt" ], "abstract": "Machine learning plays a major role in many scientific investigations. However, non-expert programmers may struggle to implement the elaborate pipelines necessary to build highly accurate and generalizable models. We introduce here a new R package, exprso, as an intuitive machine learning suite designed specifically for non-expert programmers. Built primarily for the classification of high-dimensional data, exprso uses an object-oriented framework to encapsulate a number of common analytical methods into a series of interchangeable modules. This includes modules for feature selection, classification, high-throughput parameter grid-searching, elaborate cross-validation schemes (e.g., Monte Carlo and nested cross-validation), ensemble classification, and prediction. In addition, exprso provides native support for multi-class classification through the 1-vs-all generalization of binary classifiers. In contrast to other machine learning suites, we have prioritized simplicity of use over expansiveness when designing exprso.", "keywords": [ "R", "package", "machine learning", "classification", "cross-validation", "machine learning", "supervised", "unsupervised", "genomics", "prediction" ], "content": "Introduction\n\nSupervised machine learning has an increasingly important role in biological studies. However, the sheer complexity of classification pipelines poses a significant barrier to expert biologists unfamiliar with the intricacies of machine learning. Moreover, many biologists lack the time or technical skills necessary to establish their own classification pipelines. Here we discuss the exprso package, a framework for the rapid implementation of high-throughput classification, tailored specifically for use with high-dimensional data. As such, this package aims to empower investigators to execute state-of-the-art binary and multi-class classification, including deep learning, with minimal programming experience necessary.\n\nAlthough R offers a tremendous number of high-quality classification packages, there exists only a handful of fully integrated machine learning suites for R. Of these, we recognize here the caret package which offers an expansive toolkit for both classification and regression analyses1. Otherwise, we acknowledge the RWeka package which provides an API to the popular Weka machine learning suite, originally written in Java2. While these packages have a vast repertoire of functionality, we believe the exprso package has three key advantages. First, this package employs an object-oriented design that makes the software intuitive to lay programmers. In place of a few, elaborate functions that offer power at the expense of convenience, this package makes use of more, simpler functions whereby each constituent event has its own method that users can combine in tandem to create their own custom analytical pipeline.\n\nSecond, this package exposes carefully crafted modules which simplify several high-throughput classification pipelines. Single functions, coupled with special argument handlers, manage sophisticated pipelines such as high-throughput parameter grid-searching, Monte Carlo cross-validation3, and nested cross-validation4. Moreover, users can embed these high-throughput modules (e.g., parameter grid-searching) within other modules (e.g., Monte Carlo cross-validation), allowing for infinite possibility. In addition, this package provides an automated way to build ensemble classifiers from the results of these high-throughput modules.\n\nThird, this package prioritizes multi-class classification by generalizing binary classification methods to a multiclass context. Specifically, this package automatically executes 1-vs-all classification and prediction whenever working with a dataset that contains multiple class labels. In addition, this package provides a specialized high-throughput module for 1-vs-all classification with individual 1-vs-all feature selection, an alternative to conventional multi-class classification which has been reported to improve results, at least in the setting of 1-vs-1 multi-class support vector machines5.\n\nWhile we acknowledge that the premier machine learning suites, like caret, may surpass our package in the breadth of their functionality, we do not intend to replace this tool. Rather, we developed exprso as an adjunct, or alternative, tailored specifically to those with limited programming experience, especially biologists working with high-dimensional data. That said, we hope that even some expert programmers may find value in this software tool.\n\n\nMethods\n\nThis package uses an object-oriented framework for classification. In this paradigm, every unique task, such as data splitting (i.e., creating the training and validation sets), feature selection, and classifier construction, has its own associated function, called a method. These methods typically work as wrappers for other R packages, structured so that the objects returned by one method will feed seamlessly into the next method.\n\nIn other words, each method represents one of a number of analytical modules that provides the user with stackable and interchangeable data processing tools. Examples of these methods include wrappers for popular feature selection methods (e.g., analysis of variance (ANOVA), recursive feature elimination6,7, empiric Bayes statistic8, minimum redundancy maximum relevancy (mRMR)9, and more) as well as numerous classification methods (e.g., support vector machines (SVM)10, neural networks11, deep neural networks12, random forests13, and more).\n\nWe have adopted a nomenclature to help organize the methods available in this package. In this scheme, most functions have a few letters in the beginning of their name to designate their general utility. Below, we include a brief description of these function prefixes along with a flow diagram of the available methods.\n\narray: Modules that import data stored as a data.frame, ExpressionSet object, or local text file.\n\nmod: Modules that modify the imported data prior to classification.\n\nsplit: Modules that split these data into training and validation (or test) sets.\n\nfs: Modules that perform feature selection.\n\nbuild: Modules that build classifiers and classifier ensembles.\n\npredict: Modules that deploy classifiers and classifier ensembles.\n\ncalc: Modules that calculate classifier performance, including area under the receiver operating characteristic (ROC) curve (AUC).\n\npl: Modules that manage elaborate classification pipelines, including high-throughput parameter gridsearches, Monte Carlo cross-validation, and nested cross-validation.\n\npipe: Modules that filter the classification pipeline results.\n\nElements colored grey exist outside of this package and instead refer to natively compatible components from the GEOquery14 and Biobase15 packages.\n\nWe refer the reader to the package vignette, “An Introduction to the exprso Package,” hosted with the package on the Comprehensive R Archive Network (CRAN), for a detailed description of object-oriented framework and methods used in this package16.\n\nSpecific computer hardware requirements will depend on the dimensions of the dataset under study, the methods deployed on that dataset, and the extent of any high-throughput analyses used. For the most part, however, a standard laptop computer with the latest version of R installed will handle most applications of the exprso package.\n\n\nUse cases\n\nTo showcase this package, we make use of the publicly available hallmark Golub 1999 dataset to differentiate acute lymphocytic leukemia (ALL) from acute myelogenous leukemia (AML) based on gene expression as measured by microarray technology17. We begin by importing this dataset as an ExpressionSet object from the package GolubEsets (version 1.16.0)18. Then, using the arrayExprs function, we load the ExpressionSet object into exprso. Next, using the modFilter, modTransform, and modNormalize methods, we threshold filter, log2 transform, and standardize the data, respectively, reproducing the pre-processing steps taken by the original investigators19.\n\nTo keep the code clear and concise, we make use of the %>% function notation from the magrittr package20. In short, this function passes the result from the previous function call to the first argument of the next function, an action colloquially known as piping.\n\n\n\nThen, using the splitSample method, one of the split methods shown in the above diagram, we partition the data into a training and a test set through random sampling without replacement. Next, we perform a series of feature selection methods on the extracted training set. Through the fs modules fsStats and fsPrcomp, we pass the top 50 features as selected by the Student’s t-test through dimension reduction by principal components analysis (PCA).\n\n\n\nWith feature selection complete, we can construct a classifier. For this example, we use the buildSVM method to train a linear kernel support vector machine (SVM) (with default parameters) using the top 5 principal components. Then, we deploy the trained machine on the test set from above. Note that through the objectoriented framework, each feature selection event, including the rules for dimension reduction by PCA, gets passed along automatically until classifier prediction. This ensures that the test set always undergoes the same feature selection history as the training set. The calcStats function allows us to calculate classifier performance as sensitivity, specificity, accuracy, or area under the curve (AUC)21,22.\n\n\n\nWhen constructing a classifier using a build module, we can only specify one set of parameters at a time. However, investigators often want to test models across a vast range of parameters. For this reason, we provide methods like plGrid to automate high-throughput parameter grid-searches. These methods not only wrap classifier construction, but classifier deployment as well. In addition, they accept a fold argument to toggle leave-one-out or v-fold cross-validation.\n\nBelow, we show a simple example of parameter grid-searching, whereby the top 3, 5, and 10 principal components, as established above, get used as a substrate for the construction of linear and radial kernel SVMs with costs of 1, 101, and 1001. In addition, we calculate a biased 10-fold cross-validation accuracy to help guide our choice of the final model parameters. Take note that we call this accuracy biased because we are performing cross-validation on a dataset that has already undergone feature selection. Although this approach gives a poor assessment of absolute classifier performance23, it may still have value in helping to guide parameter selection in a statistically valid manner. As an alternative to this biased cross-validation accuracy, users can instead call the plNested method in which feature selection is performed anew with each data split that occurs during the leave-one-out or v-fold cross-validation.\n\n\n\nFinally, we show an example for the plMonteCarlo method, an implementation of Monte Carlo cross-validation. Compared to the plGrid method which iteratively builds and deploys classifiers on a validation (or test) set, plMonteCarlo wraps multiple iterations of data splitting, feature selection, and parameter grid-searching. The final result therefore contains the classifier performances as measured on a number of bootstraps carved out from the initial dataset. Argument handler functions help organize the arguments supplied to the split, feature selection, and high-throughput methods managed during the plMonteCarlo method call.\n\nTake note that when using the Monte Carlo cross-validation method (or any of the other pl modules), the user may iterate over any classification method provided by exprso, not only buildSVM. This includes the buildDNN method for deep neural networks as implemented via h2o12. Also note that the user can embed other cross-validation methods, such as another Monte Carlo or nested method, within the cross-validation method call, allowing for endless combinatory possibility.\n\nIn the first section of the code below, we define the argument handler functions for the plMonteCarlo call. As suggested by their names, the ctrlSplitSet, ctrlFeatureSelect, and ctrlGridSearch handlers manage arguments to data splitting, feature selection, and high-throughput grid-searching, respectively. In this example, we set up arguments to split the unaltered training set through random sampling with replacement, perform the two-step feature selection process from above, and run a high-throughput parameter grid-search with biased cross-validation. The unaltered dataset is processed in this way 10 times, as directed by argument B.\n\n\n\nAs an adjunct to this bootstrapping pipeline, the user can apply these results to build a classifier ensemble using the best classifier from each bootstrap, then deploy that classifier on the withheld test set. Analogous to how random forests will deploy an ensemble of decision trees24, this method, which we dub “random plains”, will deploy an ensemble of SVMs.\n\n\n\nBeyond those mentioned here, this package also includes methods for integrating unsupervised machine learning (i.e., clustering) into classification pipelines. In addition, exprso contains high-throughput methods specialized for multi-class classification. We refer the reader to the package vignettes, “An Introduction to the exprso Package” and “Advanced Topics for the exprso Package”, both hosted with the package on the Comprehensive R Archive Network (CRAN), for a detailed description of all methods included in this package16.\n\n\nSummary\n\nHere we introduce the R package exprso, a machine learning suite tailored specifically to working with high-dimensional data. Unlike other machine learning suites, we have prioritized simplicity of use over expansiveness. By developing this package in an object-oriented framework, we provide a fully interchangeable and modular programming interface that allows for the rapid implementation of binary and multi-class classification pipelines. We have included in this framework functions for executing some of most popular feature selection methods and classification algorithms. In addition, exprso also contains a number of modules that facilitate classification with high-throughput parameter grid-searching in conjunction with sophisticated crossvalidation schemes. Owing to its ease-of-use and extensive documentation, we hope exprso will serve as an indispensable resource, especially to scientific investigators with limited prior programming experience.\n\n\nSoftware availability\n\nSoftware available from: http://cran.r-project.org/web/packages/exprso/\n\nLatest source code: http://github.com/tpq/exprso\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.16206325\n\nSoftware license: GNU General Public License, version 2.", "appendix": "Author contributions\n\n\n\nTQ designed and implemented the tool, applied the tool to the use case, and drafted the article. DT and SG helped design the tool and drafted the article. DT contributed code and performed extensive beta testing.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nKuhn M: Building predictive models in r using the caret package. J Stat Softw. ISSN 1548-7660. 2008; 28(1): 1–26. Publisher Full Text\n\nHornik K, Buchta C, Zeileis A: Open-source machine learning: R meets Weka. Computation Stat. ISSN 0943-4062, 1613-9658. 2008; 24(2): 225–232. Publisher Full Text\n\nPicard RR, Cook RD: Cross-Validation of Regression Models. J Am Stat Assoc. ISSN 0162-1459, 1537-274X. 1984; 79(387): 575–583. Publisher Full Text\n\nVarma S, Simon R: Bias in error estimation when using cross-validation for model selection. BMC Bioinformatics. ISSN 1471-2105. 2006; 7: 91. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuang PX, Fisher RB: Individual feature selection in each One-versus-One classifier improves multi-class SVM performance. Reference Source\n\nGuyon I, Weston J, Barnhill S, et al.: Gene Selection for Cancer Classification using Support Vector Machines. Machine Learning. ISSN 0885-6125, 1573-0565. 2002; 46(1–3): 389–422. Publisher Full Text\n\nJohannes M: pathClass: Classification using biological pathways as prior knowledge. R package version 0.9.4. 2013. Reference Source\n\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDe Jay N, Papillon-Cavanagh S, Olsen C, et al.: mRMRe: an R package for parallelized mRMR ensemble feature selection. Bioinformatics. ISSN 1367-4803, 1460-2059. 2013; 29(18): 2365–2368. PubMed Abstract \| Publisher Full Text\n\nMeyer D, Dimitriadou E, Hornik K, et al.: e1071: Misc Functions of the Department of Statistics, Probability Theory Group (Formerly: E1071). TU Wien. R package version 1.6-7. 2015. Reference Source\n\nVenables WN, Ripley BD: Modern Applied Statistics with S. Springer, New York, fourth edition. ISBN 0-387-95457-0. 2002. Reference Source\n\nAiello S, Kraljevic T, Maj P, et al.: h2o: R Interface for H2O. R package version 3.10.0.6. 2016. Reference Source\n\nLiaw A, Wiener M: Classification and regression by randomforest. R News. 2002; 2(3): 18–22. Reference Source\n\nDavis S, Meltzer PS: GEOquery: a bridge between the Gene Expression Omnibus (GEO) and Bioconductor. Bioinformatics. 2007; 23(14): 1846–1847. PubMed Abstract \| Publisher Full Text\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–121. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nQuinn T: exprso: Rapid Implementation of Machine Learning Algorithms for Genomic Data. R package version 0.1.7. 2016. Reference Source\n\nGolub TR, Slonim DK, Tamayo P, et al.: Molecular classification of cancer: class discovery and class prediction by gene expression monitoring. Science. ISSN 0036-8075. 1999; 286(5439): 531–537. PubMed Abstract \| Publisher Full Text\n\nGolub T: golubEsets: exprSets for golub leukemia data. R package version 1.12.0. Reference Source\n\nDeb K, Raji Reddy A: Reliable classification of two-class cancer data using evolutionary algorithms. BioSystems. ISSN 0303-2647. 2003; 72(1–2): 111–129. PubMed Abstract \| Publisher Full Text\n\nBache SM, Wickham H: magrittr: A Forward-Pipe Operator for R. 2014. Reference Source\n\nBradley AP: The use of the area under the ROC curve in the evaluation of machine learning algorithms. Pattern Recognition. ISSN 0031-3203. 1997; 30(7): 1145–1159. Publisher Full Text\n\nSing T, Sander O, Beerenwinkel N, et al.: ROCR: visualizing classifier performance in R. Bioinformatics. 2005; 21(20): 3940–1. PubMed Abstract \| Publisher Full Text\n\nSimon R, Radmacher MD, Dobbin K, et al.: Pitfalls in the use of DNA microarray data for diagnostic and prognostic classification. J Natl Cancer Inst. ISSN 0027-8874, 1460-2105. 2003; 95(1): 14–18. PubMed Abstract \| Publisher Full Text\n\nBreiman L: Random Forests. Mach Learn. ISSN 0885-6125, 1573-0565. 2001; 45(1): 5–32. Publisher Full Text\n\nQuinn T: tpq/exprso: exprso-0.1.7. Zenodo. 2016. Publisher Full Text" }	[ { "id": "18380", "date": "09 Dec 2016", "name": "Dariusz Plewczynski", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a short software tool article presenting a new R package for implementing machine learning pipelines. According to the authors it is targeted specifically at non-expert programmers analyzing high-dimensional biological data. The article briefly describes design goals and implementation details of the package, and then provides an example of building full machine learning pipeline for well-known microarray data set.\n\nThe main contribution of this work lies in the prepared software and not in the accompanying manuscript. Because of this in the article there are no clear hypotheses nor conclusions. The software package does not provide any novel algorithms or functionalities. It is conceived as a wrapper which should make existing methods more accessible. This goal is of practical rather than scientific nature. Ultimately, the usefulness of the package can only be confirmed by its wider adoption by the users. This situation makes it hard to write a conclusive review.\n\nI agree with the motivation behind this software. R package infrastructure can be indeed confusing and it is notoriously hard to navigate through it for a beginner programmer. I find the interface adopted by exprso package relatively clean and unambiguous. The way the evaluation methods are implemented is consistent with best machine learning practices. Eventual success of this package depends on how well it will be integrated with other popular packages. I hope that the authors will have the resources for further development of exprso.\n\nBelow I present more detailed comments for the authors:\n\ncaret is mentioned as the R package with similar goals to exprso. I would find a more detailed comparison between these two package useful, both in terms of general design and specific use cases.\n\nIt is commendable when newly created packages integrate with existing infrastructure. As I understand, exprso has some limited integration with GEOquery and Biobase packages allowing easy data import. However, after data is loaded all operations use special ExprsArray objects, distinct from native R types such as DataFrame or Matrix. I understand this design choice, but I have to note that it limits interoperability. Should the additional processing in the middle of exprso pipeline be required, data needs to be converted between formats.\n\nI find the design in which transformations applied on the training set are automatically applied on the testing set controversial. It obscures the pipeline and may not be intuitive for beginner programmers. Moreover, sometimes transformations of the testing set differ slightly from the transformations of the training set.\n\nIt is not obvious to me why simple train-test split is implemented as \"split\" module and cross-validation as \"pl\" module. There are not very much different. The way cross-validation is currently implemented does not allow detailed control over individual folds, which is sometimes useful.", "responses": [] }, { "id": "20319", "date": "17 Feb 2017", "name": "Henry Löffler-Wirth", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present a new tool integrating established algorithms into one R package. This tool is intended to provide access to state-of-the-art statistical analysis to users with limited programming experience. As the manuscript is rather a package vignette than an independent article, I focus my review mainly on the software and usability.\n\nIn general, the intention of providing comprehensive analysis options for non-experts is desirable, however a bit overachieving. In particular, I have several years of programming experience in R, but I was not able to apply the presented methods to another (multiclass) data with acceptable effort of time. Non-expert programmers, as biologists or doctors, also will not be able to correctly use the software in its present form.\n\nI therefore recommend extensive improvement of usability, user guidance and error feedback. I also acknowledge how challenging implementation of such tools is and encourage the authors to continue development of “exprso”.\n\nParticular comments:\n\nThe arrayExps-function should accept also standard matrix containing expression values, the groups may then be given as character vector. Handling of most functions can be improved by providing standard parameter values, e.g:\n\nin arrayExps (may simply include all groups) percent.include in splitSampe (66%) and so on\n\nHelp the users to find errors in their function calls by giving appropriate warning/error messages. E.g. “fsStats cannot be applied in multi-class studies”. No non-programmer will know what an “inherited method” is. Addressing the same point: fsANOVA returned with an error not clear to me (my data includes 4 groups):\n\"contrasts can be applied only to factors with 2 or more levels\"\nI found no information about this in the help file of the fs-methods. In general, documentation should be split into one individual file for each function. In the manuscript and the package vignette, the authors provide an overview figure of all functions implemented in the package. I suggest revision of this figure: a more systematic flowchart layout of the functions graph would help to find the starting point (GEO, local file, R matrix) and to follow the analysis workflow. Top-left part of the figure is not clear to me.", "responses": [ { "c_id": "3249", "date": "14 Dec 2017", "name": "Thomas Quinn", "role": "Author Response", "response": "Dear Henry,Thank you so much for taking the time to perform a detailed and critical review of the software. I regret that professional obligations have delayed me from addressing your feedback in a timely manner. However, I am pleased to say that I have heavily revised the exprso package, incorporating most, if not all, of your suggestions.Key changes include:* Easier data import with the `exprso` function which imports data in x, y format* Software now supports continuous outcome prediction (when importing data via `exprso`)* More default values (e.g., split modules) to simplify user experience* Created custom errors for every split, feature selection, build function. Most other functions now have custom errors as well* Documentation is split up by unique function. ?mod, ?split, ?fs, ?build, etc. all open a table of contents that overviews the unique functions available* Figure 1 simplified with data sources clearly labeled in black* Manuscript adjusted to reflect other changesAlso, you should not encounter any error with mutli-class classification when importing data using the new `exprso` function.(PS: I am aware of a trivial warning in this version that is triggered by predict. It is already fixed in the developmental branch on GitHub).Cheers,Thom" } ] } ]	1	https://f1000research.com/articles/5-2588
https://f1000research.com/articles/6-2087/v1	04 Dec 17	{ "type": "Case Report", "title": "Case Report: Propranolol increases the therapeutic response to temozolomide in a patient with metastatic paraganglioma", "authors": [ "Miguel-Angel Díaz-Castellanos", "Karina Villar Gómez de las Heras", "Tamara Díaz-Redondo", "Encarnación González-Flores", "Virginia Albiñana", "Luisa-María Botella", "Miguel-Angel Díaz-Castellanos", "Karina Villar Gómez de las Heras", "Tamara Díaz-Redondo", "Encarnación González-Flores", "Virginia Albiñana" ], "abstract": "This case report presents the clinical evolution and management of a patient with a hereditary paraganglioma syndrome. This disease is characterized by rare tumors of neural crest origin that are symmetrically distributed along the paravertebral axis from the base of the skull and neck to the pelvis. In addition, these patients may develop renal cancer, gastrointestinal stromal tumors, pituitary adenomas, and bone metastasis in some cases. To date no successful therapeutic treatment has been reported. Total resection with postoperative radiotherapy and chemotherapy have been advocated, especially for the multiple metastasis. Here we show how the combination of high doses of the beta blocker propranolol (3 mg/Kg/day) and the DNA intercalating agent, temozolomide, has been successful in the treatment of a SDHA metastatic paraganglioma.", "keywords": [ "Paraganglioma", "rare oncologic diseases", "repurposing drugs", "pseudohypoxic cancer syndromes" ], "content": "Introduction\n\nHereditary paraganglioma and pheochromocytoma syndromes (HPP) are characterized by rare tumors called pheochromocytomas (PHEOs) and paragangliomas (PGLs). These are highly vascularized catecholamine-secreting tumors of neural crest origin (chromaffin cells derived from the adrenal medulla – pheochromocytoma – or extra-adrenal paraganglia - paragangliomas), distributed symmetrically along the paravertebral axis. Although tumors are usually histologically benign, most of them hyper-secrete catecholamines, causing high cardiovascular morbidity and mortality1, and symptoms related to mass effect. If left untreated, a subset of these tumors will metastasize in bones, lungs, liver or lymph nodes2. These patients can develop other rare tumors too, such as renal cancer and gastrointestinal stromal tumors. However, tumorigenesis is rare in the first decade of life.\n\nHPP syndromes are due to pathogenic variants of genes, SDHx genes, a group of multiple nuclear genes encoding subunits of the succinate dehydrogenase (SDH) enzyme complex. Similarly to other autosomal-dominant hereditary cancer disease - von Hippel-Lindau (VHL) -paraganglioma–pheochromocytoma (PGL/PCC) syndrome is related to the hypoxia pathway3. Hypoxia-inducible factor 1 (HIF1) regulates the cellular response according to oxygen levels. HIF is a heterodimer of a HIF1-α subunit, regulated by oxygenation and a β subunit. Under normal oxygen conditions, HIF1-α degradation is promoted by the E3 ubiquitin ligase pVHL (von Hippel Lindau factor). In lower oxygen cell environments, VHL recognition of HIF-1α is decreased, allowing HIF1 to promote cellular survival and growth4. In cancer, the hypoxia pathway is switched by the tumoral cells leading to the tumor growth5. Pseudo-hypoxic states are those leading to the hypoxia-pathway gene expression, under normoxic conditions.\n\nGermline mutations in the onco-suppressor gene, called Vhl leads to the autosomal-dominant hereditary cancer disease VHL. VHL disease is clinically characterized by retinal and CNS hemangioblastomas, RCC (renal clear cell carcinoma), PCC, neuroendocrine pancreatic tumors and pancreatic cystadenomas, endolymphatic sac tumors and broad ligament cystadenomas. Closely related to this disease are the mutations involving the succinate dehydrogenase genes (SDHx genes)6. The lack of function of SDHx genes leads to an inhibition of prolyl hydroxylases by alpha-ketoglutarate. These hydroxylases “target” HIF1/2-α by specific hydroxylation to be recognized and bound by VHL. In this way, hydroxylases cannot help in the degradation of HIF, and a state of pseudo-hypoxia7 is created. Thus, pseudo-hypoxic states display similar hypoxia-pathway triggered gene expression, but under normoxic conditions.\n\nSurveillance recommendations for both diseases (VHL disease and PHEO/PGL)2 are very similar. It is important to suspect, localize and resect these tumors.\n\nThe present case presents the clinical evolution and management of a patient with a hereditary paraganglioma syndrome. The combination of high doses of the beta blocker propranolol (3 mg/Kg/day) and the DNA intercalating agent, temozolomide, was been successful in the treatment of the SDHA metastatic paraganglioma.\n\n\nCase description\n\nA 53 year old man, with personal and family history of hypertension, was diagnosed with a 4 cm tumoral mass at costovertebral D6–D7 with a 21 max SUV, during a routine check-up in March 2010. The only manifestation of disease was elevated blood pressure. He was being treated at this time with irbesartan/hydrochlorothiazide (1250mg/12.5mg, respectively). After surgery, the pathology diagnosis was of PGL with a Ki67 around 5-8%, and synaptophysin and chromogranin positive. Genetic analysis found a mutation in the SDHA gene, leading to a heterozygous missense p.Ser445Leu change, defined as pathogenic8. The patient remained without treatment and symptom-free until August 2014, when a back pain appeared around the D10 region, with progressive worsening. In October, as the pain persisted, he underwent MRI and 18F-DG-PET scan and was diagnosed with several metastasis at: D6–D7, right acetabulum (3cm); vertebral hemi-body D10 (3cm); vertebral body of D12 (2cm); and left iliac crest (1cm). A scintigraphy with Indium-111 pentetreotide suggested metastatic disease of probably neuroendocrine origin. D10 lesion biopsy confirmed the diagnosis of PGL metastasis with a Ki67 around 15–20% (Figure 1A). The patient was treated with doxazosin (8 mg/day) and bisoprolol (10 mg/day).\n\n(A) In October 2014, several metastasis at D6–D7, right acetabulum (3cm); vertebral hemi-body D10 (3cm); vertebral body of D12 (2cm); and left iliac crest (1cm) were observed. (B) 4 months later, a PET-TAC showed a metabolic stabilization of the disease, but with a slight growth of lesions according to MRI. (C) During the following 11 months, the patient remained stable without disease progression. (D) In May 2016, a 18 FDG-PET-TAC revealed an important metastatic dissemination all over the skeleton, showing more than 40 small new lesions (<2 cm), and with scarce or null Octreoscan and 123I-MIBG uptake. (E) In December 2016, most of lesions had disappeared according to PET-TAC. (F) In July 2017, a new PET supported the previous results, showing the remaining two lesions but with maximum SUV lower. (G) In October 2017, 4 months since the last dose of temozolomide, and propranolol as the only treatment (240mg/day), the disease remains controlled according to this last PET-TAC.\n\nIn November 2014, the patient started on systemic treatment with denosumab (120 mg every 28 days) and lanreotide (120 mg every 14 days). After 4 months, a PET-TAC showed a metabolic stabilization of the disease, but with a slight growth of lesions according to MRI (Figure 1B). D6 and D10 lesions were then treated by radiotherapy (Cyberknife). To this purpose, in March/April 2015 the patient underwent SBRT with CiberKnife (24 Gy in D10, 15 Gy in D12 and 35 Gy in D6–7), and the pain disappeared. After 6 months, the D7 and D12 lesions were also irradiated and completed SBRT with CiberKnife (24 Gy in left iliac bone and 24 Gy to right acetabulum).\n\nResponse to radiotherapy was good, showing stability of lesions and decrease of max SUV. No new lesions appeared. In June 2015, a thermoablation and right acetabulum metastasis cementation was performed with good results.\n\nAt this moment, the anti-hypertensive bisoprolol was changed to propranolol(120mg/day), due to preliminary reports of propranolol’s good results in VHL tumors9. During the following 11 months, the patient remained stable without disease progression (Figure 1C). Lanreotide dose was decreased to 120 mg every 3 weeks, due to side effects. Side effects included nausea, vomits, diarrhea and hyperglycaemia.\n\nIn May 2016, metanephrines and chromogranin were raised above normal levels, as follows: Chromogranin A, 106 ng/mL (normal level, <93); urine fractionated Norepinephrine, 196 mcg/24h (normal levels, 15–80); urine fractionated normetanephrine, 1316 mcg/24 h (normal levels, 128-484); urine total metanephephrine, 1394 mcg/24h (normal levels, 220-680). A 18FDG-PET-TAC revealed an important metastatic dissemination all over the skeleton, showing more than 40 small new lesions less than 2 cm, and with scarce or null Octreoscan and 123I-MIBG uptake (Figure 1D).\n\nFrom this moment on, a combined treatment of temozolomide (75 mg/m² for 21 days every 28 days) and an increase of propranolol up to 3 mg/kg/day (240 mg/day) was prescribed. After the first cycle of temozolomide, results were evaluated in August 2016 at National Institutes of Health, where the previously detected metastatic dissemination was confirmed, although catecholamines and chromogranin levels were decreasing compared with the previous measures. Treatment was kept the same for 6 cycles of temozolomide. In December 2016, most of lesions had disappeared according to PET-TAC (Figure 1E). The two remaining lesions had maximum SUV significantly decreased. In January 2017, lanreotide was retired due to adverse side effects (as before).\n\nIn July 2017, a new PET supported the previous results, showing the remaining two lesions but with maximum SUV lower (Figure 1F). Temozolomide was withdrawn because of side effects (lymphopenia and thrombopenia). In October 2017, 4 months since the last dose of temozolomide, and propranolol as the only treatment (240mg/day), the disease remains controlled according to the last PET-TAC (F18-FDG) (Figure 1G). This imaging study suggests the presence of two neoplastic bone lesions in the dorsal spine and right acetabulum; regarding the previous examination (July 2017), the lesions described in D6 and acetabulum persist without major changes, whereas that of vertebra D10 has disappeared.\n\n\nDiscussion\n\nThe present case report describes an impressive clinical and metabolic improvement of a patient suffering from a metastatic paraganglioma after using a combination of temozolomide and propranolol at high doses. The improvement has been kept even after 7 months of lanreotide removal (usually used as standard treatment).\n\nWe can only hypothesize about the reason for this spectacular result. However, there are some literature reports on the synergy between chemotherapeutic agents inducing DNA damage, and therefore blocking DNA replication (as it is here the case of temozolomide) and β-blockers, which have shown antiangiogenic and pro-apoptotic effects in tumoral cells9,10.\n\nRecent examples of these combinations found in the literature include the action of β-blockers increasing response to different chemotherapeutic agents. Pasquier et al. used a mouse model of neuroblastoma, where β-blockers were shown to increase treatment efficacy11. Propranolol in combination with vinblastine (chemotherapeutic agent belonging to the group of vegetable alkaloids), proved to be a successful treatment for advanced angiosarcoma12, with optimal results in seven patients following a clinical trial13.Chow et al. showed the regression of a rapidly enlarging stage T2 angiosarcoma of the scalp and face, following combination therapy with propranolol hydrochloride, paclitaxel (other vegetable alkaloid), and radiotherapy14. More recently, a prospective study using propranolol for off-label treatment of patients with melanoma, confirmed a significantly inversely association with recurrence of melanoma when propranolol was used at the time of diagnosis15. We could infer that propranolol seems to potentiate the anti-angiogenic effects and antitumor efficacy of chemotherapy.\n\nFocusing our attention to propranolol- an unspecific β-blocker - as an antitumoral agent, Albiñana and coworkers16 recently published the clinical and biomarker follow-up of a clinical trial conducted in seven VHL patients with retinal hemangioblastomas. These patients had no surgical option, and the tumors remained stable in size (with reabsorption of exudates) following 120 mg propranolol treatment for 12 months. This clinical trial led recently to the European Medicines Agency’s designation of propranolol as Orphan drug for VHL disease.\n\nThe hypothesis supporting the antiangiogenic properties of propranolol relies on its proven effect on decreasing HIF-inducible transcription targets9. Propranolol would decrease HIF protein levels, and therefore, the activation of the hypoxia program developed by the hypoxia target genes, among them, angiogenic factors, such as VEGF, FGF, PDGF, EPO, and metalloproteases, activated in tumors that favor migration and dissemination of cancer cells.\n\nThe advantages of the use of propranolol and temozolomide derive mainly from the long experience as therapeutic agents. Both are old drugs, and therefore, the safety profile and side effects are well known. In this context, we can state that if they are proven effective in tumoral cases difficult to manage, the therapeutic solution may be immediate from the bench to the patient. In our case, after 4 months taking propranolol as the only medication, the improvement is maintained, which would support the use of propranolol as a drug that significantly increases the progression-free survival in absence of temozolomide. If these results are confirmed in more studies, its use would represent an advantage, allowing - once the disease is in remission - periods of time for the marrow to recover from the adverse effects of chemotherapy with the alkylating agent.\n\nThe strengths of the treatment are the results and the absence of serious side effects for the propranolol in this particular case. The limitations of using propranolol derive from the anti-hypertensive effects. Hypotensive patients should take care with the treatment, and be always under cardiologist supervision.\n\nFor these rare diseases where therapeutic options are scarce and with not very successful results, outcomes like the one reported here have to be seriously considered if we want to improve the quality and life expectancy for these - in most cases - young patients.\n\n\nConsent\n\nWritten informed consent for the publication of the patient’s clinical details and images was obtained from the patient.", "appendix": "Author contributions\n\n\n\nMD-C, KVGH, VA and L-MB wrote the paper. MD-C, TD-R and EG-F are the physicians responsible for the patient.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nLenders JW, Duh QY, Eisenhofer G, et al.: Pheochromocytoma and Paraganglioma: An Endocrine Society Clinical Practice Guideline. J Clin Endocrinol Metab. 2014; 99(6): 1915–1942. PubMed Abstract \| Publisher Full Text\n\nRednam SP, Erez A, Druker H, et al.: Von Hippel-Lindau and hereditary pheochromocytoma/paraganglioma syndromes: Clinical features, genetics, and surveillance recommendations in childhood. Clin Cancer Res. 2017; 23(12): e68–e75. PubMed Abstract \| Publisher Full Text\n\nHenegan JC Jr, Gomez CR: Heritable Cancer Syndromes Related to the Hypoxia Pathway. Front Oncol. 2016; 6: 68. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSemenza GL: Oxygen homeostasis. Wiley Interdiscip Rev Syst Biol Med. 2010; 2(3): 336–61. PubMed Abstract \| Publisher Full Text\n\nDhani N, Fyles A, Hedley D, et al.: The clinical significance of hypoxia in human cancers. Semin Nucl Med. 2015; 45(2): 110–21. PubMed Abstract \| Publisher Full Text\n\nTaïeb D, Pacak K: New Insights into the Nuclear Imaging Phenotypes of Cluster 1 Pheochromocytoma and Paraganglioma. Trends Endocrinol Metab. 2017; 28(11): 807–817. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSelak MA, Durán RV, Gottlieb E: Redox stress is not essential for the pseudo-hypoxic phenotype of succinate dehydrogenase deficient cells. Biochim Biophys Acta - Bioenerg. 2006; 1757(5–6): 567–72. PubMed Abstract \| Publisher Full Text\n\nMiettinen M, Killian JK, Wang ZF, et al.: Immunohistochemical loss of succinate dehydrogenase subunit A (SDHA) in gastrointestinal stromal tumors (GISTs) signals SDHA germline mutation. Am J Surg Pathol. 2013; 37(2): 234–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbiñana V, Villar Gómez de Las Heras K, Serrano-Heras G, et al.: Propranolol reduces viability and induces apoptosis in hemangioblastoma cells from von Hippel-Lindau patients. Orphanet J Rare Dis. 2015; 10(1): 118. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlbiñana V, Recio-Poveda L, Zarrabeitia R, et al.: Propranolol as antiangiogenic candidate for the therapy of hereditary haemorrhagic telangiectasia. Thromb Haemost. 2012; 108(1): 41–53. PubMed Abstract \| Publisher Full Text\n\nPasquier E, Street J, Pouchy C, et al.: Β-Blockers Increase Response To Chemotherapy Via Direct Antitumour and Anti-Angiogenic Mechanisms in Neuroblastoma. Br J Cancer. 2013; 108(12): 2485–94. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPasquier E, André N, Street J, et al.: Effective Management of Advanced Angiosarcoma by the Synergistic Combination of Propranolol and Vinblastine-based Metronomic Chemotherapy: A Bench to Bedside Study. EBioMedicine. 2016; 6: 87–95. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJenkins K: Propranolol in Angiosarcoma: First Major Advance in Decades. Medscape. 2017. Reference Source\n\nChow W, Amaya CN, Rains S, et al.: Growth Attenuation of Cutaneous Angiosarcoma With Propranolol-Mediated β-Blockade. JAMA dermatology. 2015; 151(11): 1226–1229. PubMed Abstract \| Publisher Full Text\n\nDe Giorgi V, Grazzini M, Benemei S, et al.: Propranolol for Off-label Treatment of Patients With Melanoma Results From a Cohort Study. JAMA Oncol. 2017. PubMed Abstract \| Publisher Full Text\n\nAlbiñana V, Escribano RMJ, Soler I, et al.: Repurposing propranolol as a drug for the treatment of retinal haemangioblastomas in von Hippel-Lindau disease. Orphanet J Rare Dis. 2017; 12(1); 122. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "30293", "date": "12 Feb 2018", "name": "Angeles Juarranz", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe present case report describes an impressive clinical improvement of a patient suffering from a metastatic paraganglioma. The case is well written and documented with images. The good news is that the treatment is a combination of temozolomide and propranolol at high doses, old drugs from which the safety profile and side effects are well known.\nIt is noteworthy that the improvement was kept even after 7 months of lanreotide removal. Lanreotide remains one of the choice drugs as standard treatment for metastatic paragangliome, although with high side effects, and only limited success.\nWhile temozolomide is commonly used for brain tumors, the novelty is the concomitant treatment with a beta blocker, propranolol. The authors explain the results based on the antiangiogenic properties of propranolol, decreasing HIF-inducible transcription targets according to previous papers published elsewhere. Propranolol would decrease HIF protein levels, and thus the activation of hypoxia target genes, among them, angiogenic factors, such as VEGF, FGF, PDGF, EPO, and metalloproteases.\nThe advantages of the use of propranolol and temozolomide derive mainly from the long experience as therapeutic agents. Both are old drugs, and therefore, the safety profile and side effects are well known. In this context, we can state that if they are proven effective in tumoral cases difficult to manage.\nFor rare diseases where therapeutic options are scarce and with not very successful results, outcomes like the one reported here have to be seriously considered and spread in literature, since patients may get an enormous benefit. On top of this, the proposed treatment is cheap, and with minimal side effects.\nHowever, this is a case report, and to be sure that the treatment really is effective, we need more knowledge derived from other cases.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] }, { "id": "30292", "date": "21 Feb 2018", "name": "Carlos Jara", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI suggest to consider this work for indexing.\nI think the subject is interesting, the presentation is appropriate and the arguments in favor of the advantage provided by propranolol are consistent. The mechanism by which this drug adds therapeutic advantage in this type of tumor remains an enigma, but I think that as a clinical case the arguments presented are sufficient.\nThe description of the case is correct and detailed. The clinical evolution is surprising and the role that propranolol can exercise together with temozolamide is promising and frankly novel. The clinical improvement and its maintenance over time are striking, particularly after the failure to previous standard therapies.\n\nAn especially positive feature of this study is to focus on a relatively rare pathology and in which the therapeutic possibilities are limited.\n\nFor a definitive evaluation of the role that propranolol may have in the treatment of these neoplasms, together with chemotherapy, it will be necessary to develop a multicenter phase III study, which, judging by the data provided by the authors, seems to be appropriate. It is of course necessary to investigate the mechanism by which propranolol potentiate the anti-angiogenic effects and antitumor efficacy of chemotherapy.\n\nSuggestions for the authors:\nAt the beginning the phrase \"Hereditary paraganglioma and pheochromocytoma syndromes (HPP) are characterized by rare tumors called pheochromocytomas (PHEOs) and paragangliomas (PGLs).\" Should be reversed by \"Hereditary paraganglioma and pheochromocytoma syndromes (HPP) are characterized by rare tumors called paragangliomas (PGLs) and pheochromocytomas (PHEOs).\n\nIn the description of the case it should be mentioned that the study carried out presumably was an 8F-DG-PET (no data is provided other than the SUV value).\n\nI would suggest modifying \"National Institutes of Health\" by its name in Spanish, mentioning the specific hospital center.\n\nI would suggest modifying the expression 'spectacular' by a more restrained one as 'striking'.\n\nIs the background of the case’s history and progression described in sufficient detail? Yes\n\nAre enough details provided of any physical examination and diagnostic tests, treatment given and outcomes? Yes\n\nIs sufficient discussion included of the importance of the findings and their relevance to future understanding of disease processes, diagnosis or treatment? Yes\n\nIs the case presented with sufficient detail to be useful for other practitioners? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2087
https://f1000research.com/articles/6-2086/v1	04 Dec 17	{ "type": "Research Article", "title": "Characteristics of no-scalpel vasectomy patients in Jakarta, Indonesia", "authors": [ "Fakhri Rahman", "Ponco Birowo", "Akmal Taher", "Nur Rasyid", "Fakhri Rahman", "Ponco Birowo", "Akmal Taher" ], "abstract": "Background: This study aims to learn the demographic and surgical characteristics of patients who underwent no-scalpel vasectomy in Indonesia and to compare patient’s demographic characteristics before and after legal pronouncement/fatwa issued by Indonesia Ulema Council in July 2012. Methods: This is a retrospective study that collected data from the vasectomy medical records of patients who underwent no-scalpel vasectomy between January 2010 and May 2017. A total of 1497 patients were included in this study. Results: The study found that patients’ age of 40-49 years old (42.8%), wives’ age of ≥ 35 years old (65.0%), having three children (34.3%), being Moslem (85.8%), high school graduated for patients (32.3%) and junior high school graduated for patients’ wives (25.7%), casual laborer (40.7%), and guided by family planning program officer (40.6%) were the most frequent characteristics found in patients undergoing no-scalpel vasectomy. There was a significant difference in certain characteristics before and after fatwa issuance, namely wives’ age, number of children, religion, patients’ and wives’ educational background and the person who guided patients to undergo vasectomy procedure. All no-scalpel vasectomy procedures were done using “Dr. Li’s three finger technique” with local infiltration anesthesia; the median length of procedure was 10 (7 – 90) minutes. This study also found that surgeon felt that more than 95% patients were easy to perform no-scalpel vasectomy procedure and this procedure had low complication rate. Conclusions: Even though there were significant proportional difference in some characteristics, this study considers that the fatwa was not the only factor that affected patients’ choice of no-scalpel vasectomy.", "keywords": [ "characteristics", "no-scalpel vasectomy" ], "content": "Introduction\n\nEven though maternal mortality ratio has decreased since 1990, maternal death due to pregnancy- and childbirth-related complications remains a major problem, especially in developing regions1. Through Sustainable Development Goals, a continuation of Millennium Development Goals, the United Nations attempted to develop a framework to fight global problems; one of them is maternal mortality2.\n\nFamily planning programs, which were first intended to reduce the growth of the population, could reduce maternity death through contraceptive services to prevent unintended and high risk pregnancy, such as being too young or old, having too many pregnancies, or having too short an interval between pregnancies. There are several contraception methods that may involve men or women separately. In Indonesia, women still have the major role as contraception users; data from the Indonesian Health Profile 2014 showed that 49.67% of female users used hormonal injection as their contraception method3.\n\nVasectomy, one contraception method, is considered the safest and most inexpensive option for male sterilization, and no-scalpel vasectomy is preferred due to its shorter operative time and lower complication rate compared to traditional surgery4–6. However, only 0.21% prefer vasectomy to their contraception method in Indonesia3. Indonesia, the largest Moslem country, is very influenced by Islam culture, and there are still controversies regarding the use of vasectomy as a contraception method among Moslems. In July 2012, Indonesia Ulema Council (Majelis Ulama Indonesia; MUI), an institution that accommodates ulema or Moslem scholars to guide and nurture Moslems in Indonesia, issued a legal pronouncement/fatwa regarding vasectomy from haram/forbidden into halal/permissible, partly due to success of recanalization/vasectomy reversal7. Even though the fatwa still receives many controversies from other Moslem institutions in Indonesia, it might gradually change an individual’s perception regarding vasectomy.\n\nCurrently, there is no data regarding demographic characteristics of patients that have undergone no-scalpel vasectomy in Indonesia. Such data could be helpful for the government to plan and evaluate family planning programs, specifically for vasectomy services. Therefore, this study aims to learn the demographic and surgical characteristics of patients who underwent no-scalpel vasectomy in Indonesia. This study also intends to investigate patient demographic characteristics before and after the fatwa issuance.\n\n\nMethods\n\nThis study protocol received ethical approval from the Faculty of Medicine, Universitas Indonesia Ethical Committee (290/UN2.F1/ETIK/2017). The data are owned by The Indonesian Association for Secure Contraception (Perkumpulan Kontrasepsi Mantap Indonesia; PKMI) and were approved for use in this study by PKMI.\n\nThis is a retrospective observational study. The data was collected from Profamilia Clinic run by PKMI, which is based in Central Jakarta City, DKI Jakarta Province, Indonesia. PKMI is a private professional association focusing on secure contraception. Data are owned by PKMI and collected from patients under the knowledge that the data may be used for future research.\n\nVariables studied in this study were as follows: Patients’ and their wives’ age; number of children; youngest child’s age; religion; patients’ and their wives’ educational background; patients’ occupation; person who guided patients to undergo vasectomy; patient’s payment method and patients’ surgical characteristics. All variables were collected from the vasectomy medical records of patients who underwent vasectomy between January 2010 and May 2017. The medical record was designed by PKMI. Patients’ age was classified according to Indonesia Demographic and Health Survey 2012: Male module (Survei Demografi dan Kesehatan 2012 Modul Pria)8. On the other hand, patients’ wife age was classified according to high risk maternal age9. Indonesia vasectomy guidelines10 state that participants must have at least two living children or if patients have only two living children, both children must be older than two years old to undergo vasectomy. Therefore, this study also presented the number of patients who had two children with one of them still under two years of age. All other variables were classified based on vasectomy medical record10.\n\nThe data were presented in a descriptive, analytical method. Categorical data were presented by absolute value and its frequency (percentage). Numerical data were presented the mean and standard deviation if the data had normal distribution, and the median and range if the data did not have normal distribution. Any missing data was accounted for and presented in this study. Analytical statistics was used to compare patients’ demographic characteristics before and after the fatwa issuance; Chi-squared or Fisher’s test was also utilized to compare qualitative variables. Data merging was done if the data did not meet Chi-squared requirements. P-value less than 0.05 was considered statistically significant. All of analysis were done using SPSS v. 23.\n\n\nResults\n\nThere were a total of 1,497 no scalpel vasectomy procedures conducted between January 2010 and May 2017. Demographic characteristics of patients could be seen in Table 1. Most of the patients (97.4%) were paid through the governmental program.\n\nJuly 2012 is when the fatwa was issued.\n\nChi-square test\n\n1Merging data of < 20 years old and 20–29 years old due to unmet Chi-squared requirement.\n\n*2Merging data of farmer and fishermen due to unmet Chi-squared requirement.\n\nFrom 368 patients who had two children, only 25 patients had a child younger than 2 years old. Among the 1,497 patients undergoing no scalpel vasectomy, there were only 989 (66.1%) procedure reports found. All patients had infiltration with 2% lidocaine as an anesthesia technique, without any premedication, and had “Dr. Li’s three finger technique” as a surgical technique to perform no-scalpel vasectomy. All procedures used simple ligation and excision of a vas segment without using electro-cauterization. Other details of procedure report from patients included in this study are shown in Table 2.\n\nThere were a few complications reported by patients following no-scalpel vasectomy, which are presented in Table 2. This study found that no semen analysis was done within 1–3 month after the no-scalpel vasectomy procedure.\n\n\nDiscussion\n\nNo-scalpel vasectomy is a vasectomy procedure that is widely used today, due to its advantages over scalpel vasectomy; it is considered the safest and most inexpensive method for male sterilization4–6. However, it only contributes to a small percentage of the contraceptive methods used in Indonesia. This might be because the majority of Indonesian men (70.4%) have never heard about men sterilization, and among men who were aware of male sterilization methods, 77.4% never considered to undergo sterilization8. There were many factors found to affect the selection of vasectomy as a contraception method, such as lack of knowledge and negative attitudes toward vasectomy among patients and providers, education level, age, occupation, number of children, spousal support and social norms11,12.\n\nIndonesia is a country with the largest Moslem population in the world. There are still controversies regarding vasectomy procedures among Moslems in Indonesia. However, in July 2012, MUI declared a fatwa changing vasectomy from haram to halal in condition due to success recanalization/vasectomy reversal7. This study compared the demographic characteristics of patients who underwent no-scalpel vasectomy before and after MUI’s fatwa issuance. This study found no significant difference in patients’ age and patients’ occupation before and after fatwa issuance. However, there was a significant difference regarding spouses’ age, number of children, preligion, patients’ and spouses’ educational background, and the person who guided patients to undergo vasectomy procedure before and after fatwa issuance.\n\nThis study found that no-scalpel vasectomy procedure was most common in the age group of 40–49 years old (42.8%), followed by age group of 30–39 years old (25.5%) and 50–59 years old (24.5%). This finding is similar to other studies, which found that most vasectomy users underwent the procedure when they were over 30 years of age, but the largest proportion was at the age of 30–39 years old11,13–16. Moreover, based on Indonesian Demographic and Health Survey 2012 (Survei Demografi dan Kesehatan 2012), among patients considering vasectomy, the largest proportion was found at the age of 30–39 years old, followed by 40–49 years old9. Regarding the wife’s age, over 35 years old was the most common age of wife of patients undergoing no-scalpel vasectomy procedures. This is explained, since pregnancies over 35 years has a high risk for women. Other studies found the wife’s age of over 30 as the age for husbands to undergo vasectomy, while the mid- to late 30s was considered “typical” for Asian couples11,13,15,17. There was significant difference in wife’s age before and after fatwa issuance. However, this is most likely due to larger missing data after fatwa issuance, which could be due to poor data collection.\n\nAmong no-scalpel vasectomy patients, most of them had three children (34.3%) when they underwent no-scalpel vasectomy procedure. There were variations regarding the number of children among the patients across geographical regions. Although in general, the vasectomy was performed when the number of children was four or more in some places, in other places two or more children were enough to encourage patients to undergo vasectomy, such in the USA or Iran11–13,15. There were significant differences regarding the number of children before and after fatwa issuance. After July 2012, there were higher proportion of patients having two children and lower proportion of patients having three or more children compared to before July 2012. This could be a sign of the success of the family planning program. However, further study is needed to support this hypothesis.\n\nIslam is the most stated religion among patients undergoing no-scalpel vasectomy in Jakarta. This is because Islam is the majority religion in Indonesia. This study found a signficant difference in patients undergoing no-scalpel vasectomy in terms of religion before and after fatwa issuance. Interestingly, however, there were no increased participation of Moslems to undergo no-scalpel vasectomy. There was a higher proportion of Christian and Buddhist patients who underwent no-scalpel vasectomy after fatwa issuance compared to before fatwa issuance. However, an increase in the proportions of both religions could not be explained.\n\nThis study also found a high proportion of no-scalpel vasectomy patients and their spouses graduated from high school, junior high school, and elementary school (patients: 32.3%, 22.4% and 22.8%; wives: 24.6%, 25.7% and 24.9%, respectively). Therefore, this study found that the majority of patients who underwent vasectomy had less than 12 years of education. Other studies showed varied education levels across regions with the majority of patients and their spouses illiterate or had low education levels in developing countries. However, in developed countries, vasectomy was more prevalent in men with high education level16–18. There were significant differences in education level for both patients and their spouses before and after July 2012. This might be due to the increased of education participation of Indonesian people, especially at junior high school and college level. This is supported by the presence of the same trend in both patient and wife education background data, which show an increase in the proportion of junior high school and college graduated. However, the government-level data regarding Indonesia’s education participation rate are currently unavailable.\n\nMost of the patients (40.6%) undergoing no-scalpel vasectomy were suggested by family planning program officers to choose no-scalpel vasectomy as their contraception method. Family planning program officers and their cadres have an important role in helping patients to decide on vasectomy19. Data from Ghana showed that most patients gained information regarding vasectomy through media and healthcare workers, whereas only small amounts of patients gained the information from family and friends15. There was a significant difference before and after July 2012 regarding source of information/guidance who encourages patients to undergo no-scalpel vasectomy. However, this was probably due to a high proportion of missing data.\n\nAll no-scalpel vasectomy procedures were performed using “Dr. Li’s three finger technique”. This technique was developed by Dr. Li Shunqiang and was performed using ringed clamp and dissecting forcep10,20. From the procedure report, this study found that doing no-scalpel vasectomy only took a relatively short amount of time (median 10 minutes; range 7–90 minutes). Other studies found a similar short operative time for no-scalpel vasectomy6,21,22. The wide range of operating times in this study might be influenced by operator’s experience. One study found that high learning curve for no-scalpel vasectomy procedure required 10–15 operations before being able to perform the procedure perfectly23. All the procedures performed on the patients in this study were performed using infiltration as the anesthesia technique. There are several anesthetic techniques beside infiltration, such as combination of infiltration and spermatic cord block and no needle jet anesthesia. One study found that combination of infiltration and spermatic cord block was the best and the most effective method for reducing pain during vasectomy4.\n\nThis study also found that “Dr. Li’s three finger technique” was an easy procedure to conduct. Most operators felt easy in performing the steps of “Dr. Li’s three finger technique” in no-scalpel vasectomy procedure. All procedures used ligation and excision (LE) without cautery. Cautery was associated with more rapid progression to severe oligospermia and fewer early vasectomy failure24,25. However, cautery has not been adapted yet into standard practice in Indonesia. Silk was the suture material mostly used for vas ligation in this study. There were various suture materials used, such as cotton and cat gut. One study showed that the usage of vicryl increased vasectomy failure three times compared to chromic cat gut. However, until today, there was no study comparing effectiveness of silk, cotton and cat gut20,26. Most of procedures used fascial interposition (FI) as an additional step when performing no-scalpel vasectomy in this study. FI can reduce the risk of occlusive failure when performing LE and was associated with decreased time to azoospermia, decreased time to severe oligospermia and reduced failure based on semen analysis22,27. The use of combination between LE and FI varied from one country to another. There were several reasons why FI was not performed in South Asian countries, such as technical difficulties, time consuming as it adds 2–4 minutes, and national standard practice didn’t include FI as a mandatory step22.\n\nThis study found only a few complications that occurred during the no-scalpel vasectomy procedures. Another study also found few complications after no-scalpel vasectomy procedures, with an overall rate of 0.32%, consisting of hematoma, bleeding, foreign body granuloma, scrotal pain, epididymitis and sinus formation28. However, there was also a probability that patients didn’t report their complication or came to another facility, due to long distance, in this study. This study also found that no semen analysis was done after the vasectomy procedures. Based on the standard practice guideline, semen analysis should be done in the first month until the third month after vasectomy has been performed10. However, low compliance might be caused by long distance and cost. Other studies reported compliance under 30%, except for one center, in Nepal for semen analysis29.\n\nThis is the first study that which describes the characteristics of patients undergoing no-scalpel vasectomy in Indonesia. Data in this study could be useful to encourage further studies, to plan and to evaluate programs related to contraception, specifically male sterilization. However, there are also limitations in this study regarding missing data. Some variables had high missing data, such as the data regarding wife’s age and sources of information/guidance. This should encourage health care providers to pay more attention to completeness of data. Such data could be useful for a variety of goals, such as program evaluation.\n\n\nConclusion\n\nDespite its limitations, this study provides a depiction of the characteristics of patients undergoing no-scalpel vasectomy in Jakarta. Even though there were significant proportional difference in some characteristics, this study considers that the fatwa was not the only factor that affects a patient’s choice of no-scalpel vasectomy. This study also found that no-scalpel vasectomy was considered an easy procedure to perform and caused minimal complications.\n\n\nData availability\n\nDataset 1. Demographic and surgical characteristics of no-scalpel vasectomy patients. doi, 10.5256/f1000research.12748.d18566930", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nAuthors would like to say thank you to The Indonesian Association for Secure Contraception (Perkumpulan Kontrasepsi Mantap Indonesia / PKMI) for their help in providing data.\n\n\nReferences\n\nWHO, UNICEF, UNFPA, et al.: Trends in maternal mortality: 1990 to 2015: estimates by WHO, UNICEF, UNFPA, World Bank Group and the United Nations Population Division. Geneva: WHO Press; 2015. Reference Source\n\nKumar S, Kumar N, Vivekadhish S: Millennium Development Goals (MDGs) to Sustainable Development Goals (SDGs): addressing unfinished agenda and strengthening sustainable development and partnership. Indian J Community Med. 2016; 41(1): 1–4. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKementerian Kesehatan: Profil Kesehatan Indonesia 2014 [Internet]. Jakarta. Yudianto, Budijanto D, Hardhana B, Soenardi TA, editors. Kementerian Kesehatan Republik Indonesia. Jakarta: Kementrian Kesehatan Republik Indonesia; 2015. Reference Source\n\nAggarwal H, Chiou RK, Siref LE, et al.: Comparative analysis of pain during anesthesia and no-scalpel vasectomy procedure among three different local anesthetic techniques. Urology. 2009; 74(1): 77–81. PubMed Abstract \| Publisher Full Text\n\nSeenu V, Hafiz A: Routine antibiotic prophylaxis is not necessary for no scalpel vasectomy. Int Urol Nephrol. 2005; 37(4): 763–5. PubMed Abstract \| Publisher Full Text\n\nCook LA, Pun A, van Vliet H, et al.: Scalpel versus no-scalpel incision for vasectomy. Cochrane Database Syst Rev. 2007; (2): CD004112. PubMed Abstract \| Publisher Full Text\n\nMuhyiddin: Fatwa MUI tentang vasektomi: tanggapan ulama dan dampaknya terhadap peningkatan medis operasi pria (MOP). Al-Ahkam. 2014; 24(1): 69–92. Publisher Full Text\n\nBKKBN, UNFPA: Survey Demografi dan Kesehatan 2012: Modul Pria [Internet]. Jakarta; 2014. Reference Source\n\nCavazos-rehg PA, Krauss MJ, Spitznagel EL, et al.: HHS Public Access. Matern Child Heal J. 2015; 19(6): 1202–11.\n\nPKMI: Panduan pelayanan vasektomi tanpa pisau untuk pelaksana pelayanan. 3rd ed. Rasyid N, editor. Jakarta: Perkumpulan Kontrasepsi Mantap Indonesia (PKMI). 2013.\n\nNurlina R: Analisis partisipasi pria sebagai akseptor KB (kondom dan vasektomi) di wilayah kerja Puskesmas Cipanas Kecamatan Cipanas Kabupaten Lebak Provinsi Banten tahun 2011. Universitas Indonesia; 2011. Reference Source\n\nKeramat A, Zarei A, Arabi M: Barriers and facilitators affecting vasectomy acceptability (a multi stages study in a sample from north eastern of Iran), 2005–2007. Asia Pacific Fam Med. 2011; 10(1): 5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShattuck D, Perry B, Packer C, et al.: A Review of 10 Years of Vasectomy Programming and Research in Low-Resource Settings. Glob Heal Sci Pract. 2016; 4(4): 647–60. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFarrokh-eslamlou H, Oshnouei S, Alinejad V: Novel restricted access to vasectomy in Iran: Addressing changing trends in vasectomy clients' characteristics over 16years in NorthWestern Iran. Contraception. 2015; 92(5): 488–93. PubMed Abstract \| Publisher Full Text\n\nHotaling JM, Patel DP, Brant WO, et al.: Demographic and socio-economic differences between men seeking infertility evaluation and those seeking surgical sterilization: from the National Survey of Family Growth. BJU Int. 2015; 116(2): 288–92. PubMed Abstract \| Publisher Full Text\n\nOwusu-asubonteng G, Dassah ET, Odoi AT, et al.: Trend, client profile and surgical features of vasectomy in Ghana. Eur J Contracept Reprod Heal Care. 2012; 17(3): 229–36. PubMed Abstract \| Publisher Full Text\n\nGarg M, Dalela D, Dalela D, et al.: Short-term morbidity following No-Scalpel Vasectomy: an assessment of clients’ perceptions by novel postcard system. Urologia. 2014; 81(3): 177–81. PubMed Abstract \| Publisher Full Text\n\nPile JM, Barone MA: Demographics of Vasectomy--USA and International. Urol Clin North Am. 2009; 36(3): 295–305. PubMed Abstract \| Publisher Full Text\n\nAnderson JE, Jamieson DJ, Warner L, et al.: Contraceptive sterilization among married adults: national data on who chooses vasectomy and tubal sterilization. Contraception. 2012; 85(6): 552–7. PubMed Abstract \| Publisher Full Text\n\nEngenderHealth: No-scalpel vasectomy: an illustrated guide for surgeon. 3rd ed. New Jersey: EngenderHealth; 2004. Reference Source\n\nDachlan I, Rochadi S: Lama tindakan dan kejadian komplikasi pada vasektomi tanpa pisau dibandingkan dengan vasektomi metoda standar. Berkala Iimu Kedokteran. 1999; 31(4): 243–7. Reference Source\n\nLabrecque M, Dufresne C, Barone MA, et al.: Vasectomy surgical techniques: a systematic review. BMC Med. 2004; 2: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nArdiana Y, Januraga PP, Karmaya M, et al.: Faktor yang berperan pada penerimaan kontrasepsi vasektomi di Kabupaten Lombok Timur (Factors that contribute to the acceptance of vasectomy as contraception option in East Lombok Regency. Public Heal Prev Med Arch. 2015; 3(2): 218–23. Reference Source\n\nSokal D, Irsula B, Chen-mok M, et al.: A comparison of vas occlusion techniques: cautery more effective than ligation and excision with fascial interposition. BMC Urol. 2004; 4(1): 12. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBarone MA, Irsula B, Chen-mok M, et al.: Effectiveness of vasectomy using cautery. BMC Urol. 2004; 4: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKotwal S, Sundaram SK, Rangaiah CS, et al.: Does the type of suture material used for ligation of the vas deferens affect vasectomy success? Eur J Contracept Reprod Heal Care. 2008; 13(1): 25–30. PubMed Abstract \| Publisher Full Text\n\nSokal D, Irsula B, Hays M, et al.: Vasectomy by ligation and excision, with or without fascial interposition: a randomized controlled trial [ISRCTN77781689]. BMC med. 2004; 2: 6. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBhuyan K, Ali I, Sarma G, et al.: No Scalpel Vasectomy (NSV) with Ligation and Excision: A Single Centre Experience. Indian J Surg. 2015; 77(Suppl 3): 1038–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLabrecque M, Pile J, Sokal D, et al.: Vasectomy surgical techniques in South and South East Asia. BMC Urol. 2005; 5: 10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRahman F, Birowo P, Taher A, et al.: Dataset 1 in: Characteristics of no-scalpel vasectomy patients in Jakarta, Indonesia. F1000Research. 2017. Data Source" }	[ { "id": "28655", "date": "12 Dec 2017", "name": "Ryan P. Smith", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is an interesting manuscript which captures some of the barriers in Indonesia preventing widespread adoption of vasectomy. The science is sound and the commentary interesting; however, in its current state, the article requires grammatical review and correction prior to publication. There are errors throughout the text beginning in the abstract. The other clinical comment is that the author's report a guideline suggesting semen analysis be performed 1 month following the procedure, this is not the case for all guidelines (AUA suggests 8-16 weeks).\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "30098", "date": "12 Feb 2018", "name": "Dominick Shattuck", "expertise": [ "Reviewer Expertise Men's Reproductive Health" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAlthough this article has many merits, the presentation and organization of the authors ideas and work render it insufficient for indexing at this time. Unfortunately, the title and the content do not match one another. Additionally, traditional research paper structure is not followed.\n\nThis paper should be held from being indexed until the structural and editorial modifications are completed, reviewed and approved.\n\nThe title of this paper suggests that it is simply a demographic summary of users in J, Indonesia. Upon reading the abstract, another research question, the impact of Fatwa, which would be very interesting to look at comes into play. Then throughout the paper, the authors move from the Fatwa to the demographics to other analyses. this makes for a very disjointed article that is challenging for the readers to follow.\n\nOther structural issues arise, as information about the methodology is spread around the paper and the results are have no clear flow to them. The attached PDF provides detailed comments and specific locations where the authors must revisit the writing and structure of the paper.\n\nPlease click here to see the annotated PDF.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] } ]	1	https://f1000research.com/articles/6-2086
https://f1000research.com/articles/6-2075/v1	30 Nov 17	{ "type": "Research Article", "title": "The evolution of knowledge in sericultural research as observed through a science mapping approach", "authors": [ "Giselle Zambrano-Gonzalez", "Gustavo Ramirez-Gonzalez", "Martha I. Almanza-P", "Gustavo Ramirez-Gonzalez", "Martha I. Almanza-P" ], "abstract": "Background: Sericulture, since its discovery in China, has spread to become a valued activity in a range of other countries. China remains the leading producer of silk, followed by India and other Asian countries, Europe, Brazil and Colombia. This article examines the evolution of sericultural research between 1892 and 2016, identifying the main themes and applications. Methods: The SciMat software tool and the Bibliometrix R package were used as tools for data analysis in this study, based on records from the Clarivate Analytics Web of Science and SCOPUS databases. Results: The results show that research has been growing, both in number of publications from the 1990s onwards, and in the emergence of topics closely related to the sericulture research field, a field that proves to be multi-disciplinary, exhibiting expansion and vitality. Conclusions: The information gathered will contribute greatly to the definition of relevant research strands, bearing in mind a number of significant gaps in information in this field. It will furthermore provide a better insight into the development of sericulture research over time.", "keywords": [ "sericulture", "silk", "bibliometric", "evolution", "strategic diagrams", "Bibliometrix", "SciMat", "science mapping" ], "content": "Introduction\n\nSericulture is an agro-industrial activity that involves mulberry (Morus spp) cultivation, silkworm rearing, and the collection of silk thread for the textile industry. The latter aspect has been highly exploited1,2. Sericulture is carried out mainly in rural and suburban zones. Since it is limited by the supply of mulberry leaves, a lot of plants are required near the breeding site. It is environmentally friendly because it does not generate any pollutant waste, smell or sound and can therefore coexist happily with already populated areas. Silkworm rearing is not recommended however for industrial or intensely agricultural zones that use agrochemicals, given that silkworms are seriously affected by the presence of toxic product3,4.\n\nSilkworms feed on fresh mulberry leaves (leaves and young sprouts) in which they find the nutrients and water necessary to grow and develop. While artificial diets do exist, they are used only in native breeding silkworm countries, such as Japan, because of their high cost3,5,6.\n\nMulberry was first cultivated for sericulture in Asiatic countries approximately 4,500 years ago. The practice of sericulture prompted the movement of species and varieties of mulberry plant across the continents, distributing these woody plants everywhere from temperate to tropical and subtropical climates2,7. Consequently, they do not have a precise origin. Many authors agree however that the main sites of origin include regions of China, Japan and the Himalayas8,9. Mulberries can grow on non-fertile ground, but when cultivated on rich ground, with regular irrigation, they produce a large quantity of high quality leaves.\n\nIn the textile industry, no natural product surpasses the quality of silk thread, because it possesses unique characteristics in resistance, elasticity, durability, and refinement. Silk thread can also preserve heat, absorb water, gases, and colorants3,10,11; these are characteristics appreciated by the international market due to a rising tendency to use natural products, meaning that silk is increasingly welcomed and in demand.\n\nSericulture is an enterprise in which family members can work at a handcraft level so it requires low initial investment and less staff. Likewise, sub-products derived from thread production such as silkworm chrysalis and mulberry leaf residues can be used as feed for livestock, generating additional income for families.\n\nChina is considered to be the first country where spinning and knitting of silk thread took place, according to 7,000 year old archaeological findings in Yuyao city12. The trading of silk thread permitted an extensive, permanent and active economic interchange between China and other countries in ancient times, with the “Silk Road” connecting China, Mongolia, India, Persia, Arabia, Syria, Turkey, Europe and Africa12. Nowadays, spinning and knitting of silk thread is carried out in 30 countries, generally by small farmers. The main producers are China and India, who possess more than 50% of worldwide production, followed by Japan, Korea, and Thailand. The main producers in Europe are Italy and Spain, Zambia and Uganda in Africa, while in Latin America, Brazil is the largest producer, with Bolivia and Colombia as smaller producers13,14. Main consumer countries are the United States, Italy, Japan, India, France, China, United Kingdom, Switzerland, Germany, the United Arab Emirates, South Korea, and Vietnam, mainly for use in the production of clothing and home decor pieces15.\n\nBesides in the production of silk for use in textiles, it is foreseen that sericultural research will be involved in using mulberry as a bioactive substance, able to function as medicine or food, where the silkworm is studied as a “bio-factory insect” for producing protein, thanks to transformation of the mulberry. The silk itself, due to its proteic nature, could be used to manufacture membranes, as a foodstuff, in cosmetics, or for medical and bioengineering purposes.\n\nWe sought to carry out a bibliometric analysis of research into sericulture in order to identify the principal topics related to sericulture research and their applications.\n\n\nMethods\n\nWe followed four main steps to carry out the bibliometric analysis:\n\n1. Datasets from different sources were extracted;\n\n2. The data was cleaned up;\n\n3. A particular analysis of the data was carried out, depending on the software tool used;\n\n4. Lastly, the results were analyzed and discussed.\n\nSciMAT20 and the Bibliometrix21 R package (version 3.3.3) were used as tools for data analysis in this study. Both are used widely for bibliometric analysis studies. SciMAT is widely used for management and organization22,23, virtual and remote labs24, knowledge-based systems25, marketing, and26,27 in the field of social work. Bibliometrix is commonly used in businesses, healthcare, and public administration28,29.\n\nSciMAT is based on the science mapping analysis approach presented in 20, and allows us to carry out science mapping studies within a longitudinal framework30,31. The SciMAT approach establishes four steps for analysis20:\n\n1. Detection of substructures, especially clusters of words, by means of co-word analysis over specific time periods\n\n2. Generation of cluster diagrams based on results of the first step.\n\n3. Generation and interpretation of evolution diagrams based on clusters and periods. This reveals the general evolution of the research field.\n\n4. Analysis of the different time periods, clusters, and subject areas that have evolved using the various measures of density and centrality generated by the tool.\n\nA strategic diagram represents the themes of the research field. Classification into four groups is achieved using the method proposed in 32 and 33 for equivalence index, normalized for a co-word network of keyword co-occurrence. The Simple Centres algorithm is based on 34 and the values of cluster centrality and density rank are based on 32. Based on centrality and density, the clusters can be classified into four groups:\n\n1) motor\n\n2) basic and transversal\n\n3) emerging or declining\n\n4) highly developed and isolated.\n\nAn analysis of the evolution of research themes was also undertaken to detect the general areas of evolution, based on a map.\n\nBibliometrix is an R package (version 1.8) that comes with options to import bibliographic data from SCOPUS databases. The user can perform a science mapping analysis and build data matrices for co-citation, coupling, scientific collaboration analysis, and co-word analysis.\n\nThis study made use of two of the most important databases for scientific literature, i.e. the core collections of Scopus and Web of Science (Web of Science).\n\nThe analysis was performed using SciMAT version 1.1.03, configured with the following parameters: Unit of analysis, Words (authorRole=true, sourceRole=true, addedRole=true); Kind of network, Co-occurrence; Normalization measure, Association strength; Cluster algorithm, Simple Centres (Max cluster size: 7, Min cluster size: 3); Evolution measure, Association strength; Overlapping measure, Association strength.\n\nSome 1,883 records were obtained by Scopus and a further 312 by Web of Science, related to journal articles, notes, books, book chapters, reviews, and conferences. Following the deletion of duplicated records, the total of 2,195 was reduced to a final 1,930 records to be analyzed. On preliminary analysis, 91% of these were found to be for journals, with others totalling just 9%. We adopted a generalized definition of “Sericulture” which includes several different types of work on the silkworm (B. mori), mulberry (Morus spp), silk, and its industry. Some meaningless keywords that were too general, such as study or paper, were removed.\n\nUsing Bibliometrix as an R package does not require any special configuration. However, some steps based on R commands are needed:\n\n1. Upload to R the download file in a character vector.\n\n2. Convert the object to a data frame.\n\n3. Apply the “biblioAnalysis” function that returns an object of class “bibliometrix”.\n\n4. Apply the “biblioNetwork” function with the “collaboration” parameter for the “analysis” and the “countries” parameter for the “network” variable. This creates a collaboration network that is then plotted.\n\n\nResults\n\nFigure 1 shows the amount of articles published on sericulture over time, from 1892 to 2016 (plus seven documents from 2017), for Scopus and Web of Science. The information covers a 124 year period, but 93% of records were from 1990 onwards. According to these results, it could be inferred that sericulture is an increasing area of research. Of the 1,930 records found, 531 were from the years 1892 to 2000, and 1,399 from the years 2001 to 2016.\n\nThe most representative and influential journals that published on sericulture were mostly concentrated in Japan and India, except for Biomacromolecules, which is owned by the American Chemical Society.\n\nThe Journal of Sericultural Science of Japan and Japanese Journal of Human Geography are also listed, but to date these journals are not found in internationally indexed databases (Table 1).\n\n1Journals that are not categorized in JCR and SJR\n\n2Area JCR\n\nTen authors published 21.7% of the total research articles found, touching on various topics such as mulberry cropping, silkworm rearing, silk thread collection for the textile industry, and development of sericulture in different countries (Table 2). These ten authors have an affiliation from institutions located in India.\n\nThe country with the largest research output was India, with 1,242 records, much higher than the next two, Japan with 185 and China with 182. These three countries published 81.9% of the sericulture records found, and the ten most active countries produced 90% of the research (Figure 2).\n\nThirty-one countries gave meaningful contributions to sericulture from all over the globe. By continent, Asia is represented by 14 countries, Europe by 9, America by 5, Africa by 2, and Oceania by one. There are isolated publications from 19 countries, including Brazil, Italy, Turkey, Iran, Spain, and Colombia (Figure 3). Other nations with a minimal number of records found in Web of Science include Croatia, Indonesia, Madagascar, Nepal, Pakistan, Paraguay, Romania and Russia.\n\nOnce the download references were uploaded in SciMat, a total of 2,518 keywords were extracted. Table 3 presents the top ten keywords identified. These keywords represent topics related to the concepts presented in Figure 4. This figure is based on the conceptual relationship between the keywords extracted, presented in clusters.\n\nPublications were divided into five consecutive time periods: 1892–2000; 2001–2005; 2006–2010; 2011–2013; 2014–2016. These time periods had 531, 461, 532, 225, and 181 publications, respectively (Figure 5) and we found 557, 844, 1,120, 853 and 852 keywords, respectively (Figure 6). It is important to note that the first time period covers 109 years, the largest amount of research was published in the 1990s (68%).\n\nKeywords overlapping in the map of publications on sericulture: (a) 1892–2000, (b) 2001–2005, (c) 2006–2010, (d) 2011–2013 and (e) 2014–2016.\n\nFor the 1892–2000 time period 557 keywords were found. Between 2001–2005, 261 keywords did not reappear, 296 keywords migrated and 548 new keywords were mentioned. Between 2006–2010, 401 keywords did not reappear, 443 keywords migrated and 677 new keywords were mentioned. Then, between 2011–2013, 684 keywords did not reappear, 436 keywords migrated and 417 new keywords were mentioned. Finally, between 2014–2016, 499 keywords did not reappear, 354 keywords migrated and 498 new keywords were mentioned.\n\nThe number of keywords added in each of the four later time periods (64.9%, 60.5%, 48.8% and 58.5%, respectively) shows the vitality and expansion of the research field. Meanwhile, unchanged keywords between 38.9 and 53.1% (53.1%, 52.5%, 38.9% and 41.5%, respectively) point to the existence of certain base research topics (Figure 6).\n\nThe conceptual evolution of sericultural research (Figure 7) is represented by green, red and blue colours, denoting mulberry cultivation, worm breeding, and silk, respectively. There are no gaps in the evolution of most of the topics. Seven topics (Protein, Larva BM, Silk, Silkworm, Genetic, India) have been present in the field across the five time periods analysed and may thus be considered classic themes.\n\nShows the most important elements and their development. (a) Per0:1892–2000, (b) Per1:2001–2005, (c) Per2:2006–2010, (d) Per3:2011–2013 and (e) Per4:2014–2016.\n\nThe topics (mulberry cultivation, worm breeding, and silk) exhibit a positive pattern of development because of the increasing number of subtopics within them. For instance, in the case of Fiber, it diversifies into Sericin, Genetic, and Stress in the second period (Per2). Similarly, Cocoon (Per2) comes from BM larvae (Per1) and diversifies into Preservation, India, Parasite, and Economic-Conditions. Some topics sustain the number of topics from previous periods and diversify into the next; this is the case for Microscopy (Per1) that comes from the initial period (Per0) of Cocoon and Protein and changes to Fiber and Stress in the following period (Per2) (Figure 7).\n\nTo analyze the most prominent themes in the field of sericulture research for each time period, a strategic diagram is provided (Figure 8). In each diagram, sphere size is proportional to the number of records associated with each research theme.\n\nResearch themes at different time periods according to the strategic diagrams (a) 1892–2000, (b) 2001–2005, (c) 2006–2010, (d) 2011–2013 and (e) 2014–2016.\n\nThe evolution of keywords in the last 5 years is depicted in Figure 9 and Figure 10. The relationships between research themes are presented in Figure 11.\n\nKeywords overlapping map of sericulture publications in the last five years: (a) 2012, (b) 2013, (c) 2014, (d) 2015 and (e) 2016.\n\n2012 begins with 405 keywords. In 2013, 224 keywords did not reappear, 181 keywords migrated and 278 new keywords were mentioned. In 2014, 279 keywords did not reappear, 180 keywords migrated and 299 new keywords were mentioned. In 2015, 301 keywords did not reappear, 178 keywords migrated and 238 new keywords were mentioned. Finally in 2016, 296 keywords did not reappear, 120 keywords migrated and 177 new keywords were mentioned. The addition of more than half the number of keywords in each period (60.5%, 62.4%, 57.2%, and 59.6%, respectively) and the fact that less than 45% of keywords remained the same (44.7%, 39.2%, 37.2%, and 28.8%, respectively) shows the vitality and expansion of this research field in the last five years (Figure 10).\n\nThe conceptual evolution of the field of sericulture research in the last five years (Figure 12), in which the theoretical evolution since 1892–2016 is represented by three colours exhibits cohesion; furthermore, there are no gaps in the evolution of the majority of thematic areas. Protein is the thematic area that is present across the five analyzed time periods, and is thus considered an established topic in sericultural research.\n\nShows the most important elements and their development. (a) Per0:2012, (b) Per1:2013, (c) Per2:2014, (d) Per3:2015 and (e) Per4:2016.\n\nIn the last five years, topics such as SILKWORM, Bombyx mori (Supplementary Figure S1a), MULBERRY (Supplementary Figure S1b) and SILK are mentioned. Even though in one or two time periods (in the case of Silkworm, for example) they are not found as nodes that are evidenced in the clusters, these topics have been permanent and predominant. In the case of SILK, it finishes up as a motor topic. With ECONOMIC-CONDITIONS, there are not enough registers in 2012 and 2013 for it to appear as a node, but it does appear in the Cocoon and Mulberry clusters (Supplementary Figure S2); over the last three years, there is no evidence of work on this topic. For DEVELOPING, in 2012, 2013, 2014 and 2016 there are not enough registers for it to appear as a node, but it appears in the Cocoon, Larva BM, Agriculture, and Silk clusters (Supplementary Figure S3).\n\nCOCOON (Supplementary Figure S4), INDIA (Supplementary Figure S5a) and BREEDS BM (Supplementary Figure S5b) are topics that appear in a diversity of clusters: for example COCOON appears in both Silkworm and Silk, BREEDS BM appears in Silkworm and Larva BM, and INDIA appears with Mulberry. With GENETIC, there is no evidence of nodes at any time period, but it is found within the Drug, Larva BM, Animals, Bombyx mori, and Physiology clusters (Supplementary Figure S6).\n\nTo analyze the most prominent themes in the field of sericulture in the last five years, a strategic diagram is provided (Figure 13).\n\nSericulture research themes in the last five years according to the strategic diagrams (a) 2012, (b) 2013, (c) 2014, (d) 2015 and (e) 2016.\n\n\nDiscussion\n\nThree general time periods were identified for publications:\n\n(1) Pre-1990s. Only 104 publications found, possibly because over that period publishing in annexed databases wasn’t common, and the priority was other kinds of documents such as technical reports from the centres dedicated to sericulture research and journals with no annexed databases;\n\n(2) 1990s. Publications on sericulture begin to increase and specialized journals are created;\n\n(3) Post-2000s. More than 76% of publications are from this time period, with strong growth in 2003, 2004, 2007, 2008 and 2009.\n\nThe publication dynamics in sericulture appear to be related to the history of fibres. Ever since the appearance of synthetic fibres (early XX century)16, the market has evolved thanks to the cost, ease of production, greater uniformity,17, such that they tended to push natural fibers out of the marker. However, in the last few years, natural fibres have been viewed as a sustainable option, 100% biodegradable, environmentally friendly, this has caused their use to diversify and increase18.\n\nThe journals that have published the most on the topic of sericulture have a low impact factor that can be attributed to their specialization. The appearance and indexing of specialized journals such as Silk and India Journal of Sericulture in the 1990s lead to an increase in publication output. The total number of journals that have published sericulture related topics is 528. The top 10 journals account for 62.4% of the total contributions (1,206).\n\nPublications in the last 20 years have been led by authors from India. Notably, the Central Sericultural Research and Training Institute, founded in 1961 and directed by the Ministry of Textiles of the Government of India, is actively developing the silk industry in the country, and its researchers represent the most productive authors. From the total number of authors (3,058), ten have published 21.7% of the research output (420 documents) and 83.5% (2,555) have published one or more documents.\n\nThe importance of India in the production of sericulture documents worldwide may lie in the investment the country makes in developing the silk industry, including silk centres, research institutes, specialized journals, and organized events. Publications with collaborations are evident; Japan has the most publications in collaboration with authors from other nations with 12 country links, followed by China with 10 and India with 6 links. This might suggest that collaborative work is missing between countries with an established research output and those looking at sericulture as an option for diversification and economic development, as is the case with Colombia and Brazil in South America19.\n\nGenerally, the keywords found were not linked just to one area of sericulture (mulberry cultivation, worm breeding, silk), showing that sericulture is multidisciplinary. It is worth mentioning that this research has shown that sericulture is dynamic and has faced new challenges created by environmental needs and scientific progress, as shown by keywords such as molecular, transgenic, genomic, sequence, and phylogenetic.\n\n\nConclusions\n\nA bibliometric analysis of publications between 1892 and 2016, was carried out for the field of sericulture research. It was found that most research is published in journals in China and Japan, while the most representative authors were affiliated to research centres in India. As a result, India produces 64% of publications. The research field is centred on topics related to silk, silkworm, Bombyx mori, mulberry, cocoon, India, BM breeds, economic conditions, genetic, and development. As proposed by 35, B. mori is excellent as an experimental animal for genetic and biological research, and was present in several clusters in the course of the analysis. The field is multi-disciplinary and exhibits expansion and vitality. In future work, this type of bibliometric analysis gives researchers a broader view of the research field, to better identify potential interplay of topics in future work.\n\n\nData availability\n\nDataset 1: Data obtained from Web of Science in May 2017. DOI, 10.5256/f1000research.12649.d18483736\n\nDataset 2: Data obtained from Scopus files in ris format in May 2017. DOI, 10.5256/f1000research.12649.d18483837\n\nDataset 3: Data obtained from Scopus files in BibTex format in May 2017, to be used with Bibliometrix. DOI, 10.5256/f1000research.12649.d18483938\n\nDataset 4: SCIMAT project file, to be opened in SciMAT with the processed files. DOI, 10.5256/f1000research.12649.d18521839", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nThe authors wish to thank the University of Cauca and the following research groups: the Geology, Ecology and Conservation research group (GECO), Telematic Engineering Group (GIT) and the Integrated Production Systems Research Group (SISINPRO). Also to the projects InnovAccion-Cauca and Technical Development for Organic and Innovative Silk Products Project, from the General Royalties System, and the Government of Cauca. We are especially grateful to Colin McLachlan for suggestions relating to the English text.\n\n\nSupplementary Material\n\nFigure S1. Present topics inside the clusters of the last five years.\n\nClick here to access the data.\n\nFigure S2. The keyword Economic-conditions is present in some clusters of the last five years (a) initial period (Per0: 2012), (b) second period (Per1: 2013).\n\nClick here to access the data.\n\nFigure S3. The keyword Developing is present inside the clusters of the last five years (a) initial period (Per0: 2012), (b) second period (Per1: 2013), (c) third period (Per2: 2014), (d) fifth period (Per4: 2016).\n\nClick here to access the data.\n\nFigure S4. The keyword Cocoon is present inside the clusters of the last five years (a) third period (Per2: 2014), (b) fourth period (Per3: 2015), (c) fifth period (Per4: 2016).\n\nClick here to access the data.\n\nFigure S5. Present topics in some clusters of the last five years. Topics that appear in a number of clusters include India in (a) for the second period (Per1: 2013), and Breed BM in (b) for the third period (Per2: 2014).\n\nClick here to access the data.\n\nFigure S6. The keyword Genetic present inside clusters of the last five years (a) initial period (Per0: 2012), (b) second period (Per1: 2013), (c) third period (Per2: 2014), (d) fourth period (Per3: 2015), (e) fifth period (Per4: 2016).\n\nClick here to access the data.\n\n\nReferences\n\nCifuentes C, Sohn KW: Manual técnico de sericultura: Cultivo de la morera y cría del gusano de seda en el trópico). SENA-CDTS, 1998.\n\nAruga H: Principles of Sericulture. CRC Press, 1994. Reference Source\n\nPescio F, Zunini H, Pedro BC, et al.: Sericultura: Manual para la produccion. Instituto Nacional de Tecnologia Industrial (INTI), 2006. Reference Source\n\nMunhoz REF, Bignotto TS, Pereira NC, et al.: Evaluation of the toxic effect of insecticide chlorantraniliprole on the silkworm Bombyx mori (Lepidoptera: Bombycidae). Open J Anim Sci. 2013; 3(4): 343–353. 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Technical report, Pademer, 2003. Reference Source\n\nCobo M, López-Herrera A, Herrera-Viedma E, et al.: An approach for detecting, quantifying, and visualizing the evolution of a research field: A practical application to the Fuzzy Sets Theory field. J Informetr. 2011; 5(1): 146–166. Publisher Full Text\n\nAria M, Cuccurullo C: bibliometrix: an R tool for comprehensive bibliometric analysis of scientific literature. University of Naples Federico II Naples, Italy, 2016.\n\nZupic I, Cater T: Bibliometric Methods in Management and Organization. Organ Res Methods. 2015; 18(3): 429–472. Publisher Full Text\n\nHeradio R, de la Torre L, Galan D, et al.: Virtual and remote labs in education: A bibliometric analysis. Comput Educ. 2016; 98: 14–38. Publisher Full Text\n\nCobo M, Martínez M, Gutiérrez-Salcedo M, et al.: 25years at Knowledge-Based Systems: A bibliometric analysis. Knowl Based Syst. 25th anniversary of Knowledge-Based Systems. 2015; 80: 3–13. Publisher Full Text\n\nMurgado-Armenteros EM, Gutiérrez-Salcedo M, Torres-Ruiz FJ, et al.: Analysing the conceptual evolution of qualitative marketing research through science mapping analysis. Scientometrics. 2015; 102(1): 519–557. Publisher Full Text\n\nMartínez MA, Herrera M, Contreras E, et al.: Characterizing highly cited papers in Social Work through H-Classics. Scientometrics. 2015; 102(2): 1713–1729. Publisher Full Text\n\nMartínez MA, Cobo MJ, Herrera M, et al.: Analyzing the Scientific Evolution of Social Work Using Science Mapping. Res Soc Work Pract. 2015; 25(2): 257–277. Publisher Full Text\n\nCuccurullo C, Aria M, Sarto F: Foundations and trends in performance management. A twenty-five years bibliometric analysis in business and public administration domains. Scientometrics. 2016; 108(2): 595–611. Publisher Full Text\n\nAria M, Cuccurullo C, Sarto F: Exploring healthcare governance literature: systematic review and paths for future research. MECOSAN. 2015; 61–80. Publisher Full Text\n\nGarfield E: Scientography: Mapping the tracks of science. Current Contents: Social & Behavioural Sciences. 1994; 7: 5–10. Reference Source\n\nde Solla Price D, Gürsey S: Studies in Scientometrics I Transience and Continuance in Scientific Authorship. Ciência da Informação. 1975; 4(1). Reference Source\n\nCallon M, Courtial JP, Laville F: Co-word analysis as a tool for describing the network of interactions between basic and technological research: The case of polymer chemsitry. Scientometrics. 1991; 22(1): 155–205. Publisher Full Text\n\nCourtial JP, Callon M: Indicators for the identification of strategic themes within a research programme. Scientometrics. 1991; 21(3): 447–457. Publisher Full Text\n\nCoulter N, Monarch I, Konda S: Software engineering as seen through its research literature: A study in co-word analysis. J Am Soc Inf Sci. 1998; 49(13): 1206–1223. Publisher Full Text\n\nPetkov N, Tzenov P, Petkov Z, et al.: Silkworm, Bombyx mori L. germplasm resources in Bulgaria. Technical report, National Centre for Agrarian Sciences–Sofia, Sericiltural Experiment Station–Vratza, 2006. Reference Source\n\nRamirez-Gonzalez G, Zambrano-Gonzalez G, Almanza-P MI: Dataset 1 in: The Evolution of knowledge in Sericultural Research through a science mapping approach. F1000Research. 2017. Data Source\n\nRamirez-Gonzalez G, Zambrano-Gonzalez G, Almanza-P MI: Dataset 2 in: The Evolution of knowledge in Sericultural Research through a science mapping approach. F1000Research. 2017. Data Source\n\nRamirez-Gonzalez G, Zambrano-Gonzalez G, Almanza-P MI: Dataset 3 in: The Evolution of knowledge in Sericultural Research through a science mapping approach. F1000Research. 2017. Data Source\n\nRamirez-Gonzalez G, Zambrano-Gonzalez G, Almanza-P MI: Dataset 4 in: The Evolution of knowledge in Sericultural Research through a science mapping approach. F1000Research. 2017. Data Source" }	[ { "id": "28574", "date": "20 Dec 2017", "name": "Manuel Jesus Cobo", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this paper, a bibliometric analysis of the Sericulture field has been carried out by means of science mapping analysis. To do that, SciMAT and Bibliometrix was employed. Although the paper is interesting, it must be further improved. In general, it is not well organized and it is difficult to read, and therefore, understand. Regarding the methodology, authors must to clarify the use of two software. And also, authors must to read carefully the methodology used since some concepts seem not clear. Moreover, the contribution of the paper to the field is very little. Authors do not extract relevant conclusion from the results. They just describe the figure and tables. Also, the objectives are not clear.\nIn what follows, some comments and suggestion are listed.\nThe introduction describes the sericulture research field deeply, but the objective and research questions of the paper are missing. Authors must to clarify if there are other bibliometric analyses focused on that topic, and the importance of this research.\n\nTwo software tools are used in this study: SciMAT and Bibliometrix. But, they are not properly cited. Both software have associated a publication published in important journals. For example, SciMAT is published in JASIST in 20121 and Bibliometrix is published in JOI in 20172. Authors should cite correctly the software used. Moreover, there is a reference to SciMAT, but authors confused the methodology used by SciMAT with the paper describing SciMAT. Both paper should be cited.\n\nWhy authors use SciMAT and Bibliometrix?\n\nWhat is the query used to retrieve the data?\n\nAuthors should clarify why they used two different databases to retrieve the data. If two databases are used, authors should not take into account the citations, since may vary in WoS and Scopus.\n\nAuthors perform a deduplication step over the keywords?\n\nHow Figure 4 was created?\n\nWhy the conceptual evolution is shown before the strategic diagrams? It does not make sense. Thus, Figure 8 (strategic diagrams) must be place before Figure 7 (evolution).\n\nLast paragraph of page 6 does not provide nothing interesting, since it is just describing the overlapping figure in a textual way. Authors should make an effort to show the knowledge learned.\n\nRegarding the strategic diagrams, authors just put them but any comment is added. This is the most important part of the papers, and there is any comment describing them. Also, the strategic diagrams are difficult to understand since they have a lot of themes. I recommend to set a higher threshold for the data and network in SciMAT.\n\nWhy authors make a complementary analysis of the last five years in five different periods? In this subanalysis, authors made the same errors than in the analysis of the other periods. That is, no comments (or just a few) describing the results.\n\nFinally, the discussion and conclusion must be further improved. Authors must make a great effort extracting knowledge from their results. That is, authors should not just mention the results shown in Tables and Figures, but describe and interpret the results.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "30105", "date": "02 Mar 2018", "name": "Olga Scrivner", "expertise": [ "Reviewer Expertise Data analytics", "text mining", "NLP", "visualization" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors provide a historical overview of silk cultivation, followed by a bibliometric analysis of literature ranging from 1892 to 2016. Their findings identify an increase in published research and the growing number of keywords over time. In addition to the analysis, the authors provide technical information on the software used for the analysis, namely R package Bibliometrix and SciMAT.\n\nDespite the interesting topic, the reviewer has identified several weaknesses, including the need for description of data processing and more clear interpretation of results. Exact pre-processing steps and WOS/SCOPUS query extraction are essential to ensure the reproducibility. In addition, the review of literature on bibliometric analysis is highly recommended.\nSeveral figures are not clearly described. For example, Figure 3 states \"Collaboration between countries\", whereas in the text it refers to isolated publications by country. It is also desirable to re-render figures with a better quality, if feasible.\nMore specific questions:\nTools SciMAT tool - Cluster classification - consider adding explanation to four cluster groups\n\nWhat is the reasoning behind five consecutive unequal time periods\n\nExplain Figure 6 - how it was built and how to interpret it.\n\nExpand on or rephrase the last paragraph p.6\n\nExpand on and describe the strategic diagram Fig 13\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? No\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-2075
https://f1000research.com/articles/6-2074/v1	30 Nov 17	{ "type": "Software Tool Article", "title": "Using bio.tools to generate and annotate workbench tool descriptions", "authors": [ "Kenzo-Hugo Hillion", "Ivan Kuzmin", "Anton Khodak", "Eric Rasche", "Michael Crusoe", "Hedi Peterson", "Jon Ison", "Hervé Ménager", "Ivan Kuzmin", "Anton Khodak", "Eric Rasche", "Michael Crusoe", "Hedi Peterson", "Jon Ison" ], "abstract": "Workbench and workflow systems such as Galaxy, Taverna, Chipster, or Common Workflow Language (CWL)-based frameworks, facilitate the access to bioinformatics tools in a user-friendly, scalable and reproducible way. Still, the integration of tools in such environments remains a cumbersome, time consuming and error-prone process. A major consequence is the incomplete or outdated description of tools that are often missing important information, including parameters and metadata such as publication or links to documentation. ToolDog (Tool DescriptiOn Generator) facilitates the integration of tools - which have been registered in the ELIXIR tools registry (https://bio.tools) - into workbench environments by generating tool description templates. ToolDog includes two modules. The first module analyses the source code of the bioinformatics software with language-specific plugins, and generates a skeleton for a Galaxy XML or CWL tool description. The second module is dedicated to the enrichment of the generated tool description, using metadata provided by bio.tools. This last module can also be used on its own to complete or correct existing tool descriptions with missing metadata.", "keywords": [ "bioinformatics", "tool integration", "galaxy", "common workflow language", "interoperability", "registry" ], "content": "Introduction\n\nOver the last few years, bioinformatics has played a major role in the field of biology, raising the issue of best practices in software development for the members of the bioinformatics community1–3. These practices include facilitating the discovery, deployment, and usage of tools, and several helpful solutions are available.\n\nTool discovery is facilitated by various online catalogs and registries4–6. The ELIXIR Tools and Data Services Registry, bio.tools7, describes bioinformatics software using extensive metadata descriptions, supported by the EDAM ontology8.\n\nFor software deployment, distribution systems are available9–13 that let users locally install the tools that they need in convenient, portable and reproducible ways. Workbench and workflow systems such as Galaxy14,15, Taverna16 or Chipster17 allow the execution and composition of bioinformatics tools in integrated environments which aim at improved usability, interoperability and reproducibility. Finally, the Common Workflow Language18 (CWL) is a recent project that defines a standardized and portable tool and workflow description format, usable across different platforms.\n\nAll of the above systems rely on components that provide the necessary information to describe, install, or run a specific piece of software. Gathering this information and formatting it into tractable tool descriptions is often a complex and time consuming task for developers. Indeed, it requires a deep knowledge of both the tool itself and the description format. A significant part of the metadata stored in the descriptions is, however, common to registries and workbench environments systems19, and strategies relying on a mapping between these different description formats can help avoid redundancy and mislabeling of tools Figure 1). The ReGaTE utility20 illustrates this by using tool descriptions from Galaxy to publish available services on bio.tools. Another application is to facilitate workbench environment integration, by reusing tool descriptions from registries. Here we present “ToolDog” (Tool DescriptiOn Generator), an application that enables workbench integration for tools registered in the bio.tools registry.\n\nThe objective is to integrate the bio.tools registry with workbench environments in two ways: (1) “ReGaTE”, a utility for en masse registration of services from Galaxy instances; (2) the “ToolDog” utility, to translate the description of any tool or service that is registered in bio.tools, into the format required by the existing major workbench environments.\n\nBioinformatics tools are described in various formats and levels of detail, befitting different systems and use-cases. A bio.tools entry provides tool descriptions for tool end-users, primarily for search and discovery purposes. The metadata provides a basic description including the tool type, what task it performs, the main input and output data, who created it, where it is available, and its license. This description, based on the BiotoolsSchema model, can be accessed through the bio.tools API and retrieved in JSON format. Conversely, Galaxy and CWL tool descriptions must support tool discovery, execution, and integration into homogeneous environments. This requires an extensive description of their command line syntax (or other type of API). Galaxy tool descriptions are written in XML or YAML, and the corresponding XSD is available. CWL tool descriptions are described using the YAML-based SALAD format.\n\nAll three of these tool description formats provide the possibility of specifying EDAM terms. In bio.tools this can be done directly. CWL supports these annotations through the addition of bioschemas mark-up, and Galaxy supports EDAM through specific tags mapping to its internal typing system21. The EDAM ontology helps with the description of the tools by providing a common vocabulary that includes terms to describe topics that specify which particular domains of bioinformatics the tool serves, operations that describe what the tool does, and data and formats that specify the type and format of the inputs and outputs.\n\nTool descriptions for workbench systems are expensive to create and maintain, because they require exhaustive knowledge of both the described tool, and the syntax used for the description19. Consequently, tool descriptions are sometimes incomplete or out of date. For instance, in the case of Galaxy, the analysis of the main server and the server of the Institut Pasteur22 shows that some tools are not adequately described (see Figure 2). Specifically, although most of the tools have a help section and a description, important elements such as citation information are often missing. The evolution of the Galaxy framework itself also generates a need for maintenance, through changes in the tool description format. With the recent addition of EDAM annotations tags in the format, tools had to be updated to support this new feature. The users of such graphical workbench platforms do not typically handle tool discovery and deployment tasks. Thus, detailed tool descriptions are fundamental, because they are the main source of information for the scientists who use them.\n\nDifferent approaches exist to help improve the quality of the corpus of tool descriptions. (1) Tooling facilitates the creation and validation of the tool descriptions, using Planemo23 in the case of Galaxy. (2) Community approaches such as the Intergalactic Utilities Commission design and promote best practices for the development of Galaxy tools. (3) Standardization efforts like CWL also reduce the maintenance work for tool descriptions by making them portable between different platforms.\n\nMetadata coverage for Galaxy tool descriptions from (A) the main Galaxy instance (https://usegalaxy.org) and (B) the Institut Pasteur Galaxy instance (https://galaxy.pasteur.fr). The graphs show the percentage of tools possessing various metadata types: Help: usage instructions; Description: description of the tool to be displayed in the tool menu; Citations: tool citation information using either a DOI or a BibTeX entry; H+D+C: contains a help, description and citations section; Operations: description of the EDAM operation(s) performed; Topics: description of the EDAM topics covered. The total number of tools includes those which were successfully retrieved and analyzed (672 out of 1209 on Galaxy main, 351 out of 526 on Pasteur); not all available tools were retrieved - some because they are not available in a ToolShed, and some because we chose to retrieve only the latest version of each tool and discarded the earlier ones.\n\nToolDog complements all of these approaches. It leverages the information available in bio.tools to simplify the integration of bioinformatics software into workbench environments.\n\n\nMethods\n\nToolDog is a command-line utility written in Python. It consists of two modules, which handle (1) the generation of a skeleton for the tool description, based on the analysis of the source code of the tool, and (2) the enrichment of the tool description, using the bio.tools metadata. The tool description generation pipeline (Figure 3) leverages bio.tools and includes both a module to generate a tool description using only the registry, as well as a module to enrich an existing tool description with information from the registry.\n\nFor a number of bioinformatics tools, a significant part of their description can be extracted from an analysis of the source code. The source code analysis module of ToolDog does this, currently only with python-based tools that use the argparse library for parsing command-line arguments. This module uses the argparse2tool package to retrieve the list of parameters and generate Galaxy or CWL tool description skeletons. To generate such skeletons, ToolDog runs a Docker software container that will download, install, analyze the source code, generate the tool description and then retrieve it. This strategy avoids the pollution of the local user’s environment and provides a completely pre-configured, ready-to-use installation of ToolDog.\n\nGalaxy and CWL tool descriptions, whether they were manually authored or automatically generated by source code analyses, can be improved by the description enrichment module. This retrieves additional metadata from the corresponding bio.tools entries, and fills in the missing information in the workbench tool description when available.\n\nInternally, the input tool description is parsed into an object model of the tool. The metadata from bio.tools are then mapped onto this object model, which is later exported to Galaxy or CWL formats. Parsing and export capabilities of ToolDog leverage the galaxyxml or cwlgen libraries to import and export the updated descriptions.\n\n\nResults\n\nHere we illustrate the generation of a tool description with the example of IntegronFinder24, an analysis tool dedicated to the identification of integrons in bacterial genomes. Launching ToolDog in “generation mode” on the IntegronFinder entry in the bio.tools registry allows the generation of a significant portion of the tool description (Figure 4), either in CWL or Galaxy format. Some manual modifications (corrections + additions) are still necessary to complete the tool description and to make it functional. For instance, software requirements, which specify what software needs to be installed for the tool to run correctly, cannot be automatically generated, because this information is currently not available in bio.tools. Additionally, the mapping between inputs and the generated command line, as well as between outputs and the file names they refer to is not present.\n\nIn addition to novel tool description generation, ToolDog can also perform the automated enrichment of existing tool descriptions with bio.tools metadata. To test this approach, we ran ToolDog on the tool descriptions available on the Galaxy main instance that lack EDAM annotations. All of the Galaxy descriptions from the main instance were retrieved, and mapped to bio.tools entries using the citation identifiers (DOI). The goal was to add EDAM terms describing the topic of application and the operation(s) performed by the tools. To avoid linking unrelated entries, we took a conservative approach, only mapping by default two entries when they referred to, and only to, the same publication. The results (Figure 5) show that whenever this linking can be reliably done, the enrichment can easily be performed, with a total of 217 Galaxy tool descriptions being enriched out of 224 being initially mapped to bio.tools. A detailed description of this analysis, including the original and annotated tool descriptions, is available at https://github.com/khillion/galaxyxml-analysis/annotate_usegalaxy.\n\nOut of 665 retrieved tool descriptions, 399 have a DOI and 224 of these descriptions could be mapped to a bio.tools entry. 217 tool descriptions have been successfully annotated using ToolDog (Citations: presence of tool citation information; DOI: tool citation information described using a DOI; Corresponding bio.tools: tool descriptions with a corresponding bio.tools entry retrieved using the DOI; Annotated tools: tool descriptions successfully annotated with ToolDog).\n\n\nDiscussion\n\nThe ToolDog utility allows a developer to generate new tool descriptions for tools which are compatible with the code analysis module, and reuse the metadata provided by bio.tools to enrich existing tool descriptions. There are some limitations to this approach:\n\n1. The “plugin” libraries used for code analysis are specific to the programming languages, libraries or framework used to build the command line interface. To this date, they don’t cover most of these.\n\n2. The generation of the tool descriptions through code analysis must assume certain coding practices, such as the use of specific functions to define input or output parameters, which are not uniformly adopted.\n\n3. Some of the input/output operations performed by some programs are a lot more difficult to detect through code analysis because they are typically not included in command line parsing frameworks, such web service and database queries and submissions, or in place file modifications.\n\nThe automated enrichment of existing tool descriptions provides a convenient way to improve them, especially if they lack most of the metadata provided by bio.tools. Performing this enrichment efficiently en masse, however, would require the wide adoption of an identification system for bioinformatics software. This mechanism would allow to avoid the complex and sometimes ambiguous mapping procedures based on publication identifiers we performed when testing it on the Galaxy tools. A recent update to bio.tools has added stable and unique tool identifiers, based on registered tool names, yielding persistent references to tools, for example https://bio.tools/signalp. Future work will make use of these identifiers to improve the generation of tool descriptions. For instance, linking of the bioconda and biocontainers repositories to bio.tools will enable ToolDog to generate software requirements compatible with workbench platforms25.\n\n\nConclusions\n\nDuring the last years, integration of various tools has been eased by the use of workbench systems such as Galaxy, and frameworks using the Common Workflow Language. Still, it remains time consuming and not straightforward to adapt resources to such environments. ToolDog lays the foundation for future work, that will provide a Workbench Integration Enabler for the bio.tools registry as an online service. Furthermore, integration with Planemo, the main utility to develop Galaxy and CWL tools, will be further developed in order to make the simple, bio.tools-based metadata enrichment of ToolDog available to the widest possible audience.\n\n\nData availability\n\nThe scripts and results of the analysis performed to motivate and test our approach are available at: https://github.com/khillion/galaxyxml-analysis, and are archived at the time of publication at: https://doi.org/10.5281/zenodo.103800526.\n\n\nSoftware availability\n\nThe ToolDog software is available at: https://pypi.python.org/pypi/tooldog\n\nThe source code is available at: https://github.com/bio-tools/tooldog\n\nArchived source code as at the time of publication: https://doi.org/10.5281/zenodo.103790927\n\nSoftware license: MIT License.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nELIXIR-EXCELERATE is funded by the European Commission within the Research Infrastructures Programme of Horizon 2020 [676559].\n\n\nAcknowledgments\n\nJon Ison acknowledges the support of the Danish ELIXIR Node. Kenzo-Hugo Hillion and Hervé Ménager wish to thank Fabien Mareuil, Olivia Doppelt-Azeroual, Bertrand Néron from the Institut Pasteur, as well as Daniel Blankenberg (Cleveland Clinic) and John Chilton (Galaxy Project) for their technical advice during the development. Anton Khodak wishes to thank his Google Summer of Code mentor Roman Valls Guimera (University of Melbourne), who promoted the idea of argparse2tool and supervised his internship.\n\n\nReferences\n\nArtaza H, Chue Hong N, Corpas M, et al.: Top 10 metrics for life science software good practices [version 1; referees: 2 approved]. F1000Res. 2016; 5: pii: ELIXIR-2000. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nJiménez RC, Kuzak M, Alhamdoosh M, et al.: Four simple recommendations to encourage best practices in research software [version 1; referees: 3 approved]. F1000Res. 2017; 6: pii: ELIXIR-876. 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Publisher Full Text\n\nCury J, Jové T, Touchon M, et al.: Identification and analysis of integrons and cassette arrays in bacterial genomes. Nucleic Acids Res. 2016; 44(10): 4539–4550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrüning B, Chilton J, Köster J, et al.: Practical computational reproducibility in the life sciences. bioRxiv. 2017. Publisher Full Text\n\nHillion KH, just another pesky drone: khillion/galaxyxml-analysis: v1.0.2 for F1000 submission (Version v1.0.2). Zenodo. 2017. Data Source\n\nHillion KH, just another pesky drone, Kuzmin I, et al.: bio-tools/ToolDog: v0.3.4 for F1000 submission (Version v0.3.4). Zenodo. 2017. Data Source" }	[ { "id": "28566", "date": "15 Dec 2017", "name": "Michael L. Heuer", "expertise": [ "Reviewer Expertise Bioinformatics", "big data genomics", "immunogenomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFrom the point of view of a software tool author, it is not a simple task to provide high-quality metadata and software tool descriptions. Any tooling that supports DRY (don't repeat yourself) in this regard is most welcome.\nThe authors describe a path from the bio.tools bioinformatics software registry, which uses a rich metadata schema for syntax, the EDAM ontology for semantics, and strongly written guidelines to ensure high-quality entries, to tool descriptions for the Galaxy workbench and in Common Workflow Language (CWL) for use on various workflow execution environments.\nMuch of the metadata in the tool descriptions is generated by the ToolDog utility from an entry in bio.tools, ensuring proper mapping between metadata concepts. This would be a great help when bootstrapping Galaxy and CWL support for a new software tool. The authors also describe and implement a use case for enriching existing tool descriptions.\nI am curious if there are practical benefits to enriching tool descriptions with EDAM ontology terms, in addition to quality of metadata from using well defined terms from a controlled vocabulary?\nThe source code is available at the Github link provided and is licensed MIT License as stated in the paper. I appreciate that the scripts and results of the analysis are archived as well.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "28565", "date": "18 Dec 2017", "name": "Christopher J. Fields", "expertise": [ "Reviewer Expertise Computational biology" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper presents a very nice overview on how ToolDog is used to (1) generate new tool descriptors for Galaxy and CWL from code analysis, and (2) improve documentation for current tools from the bio.tools registry. This provides a valuable service to the bioinformatics community and in particular to ensuring that tooling information is consistently described but also updatable. In my opinion this should be accepted, with some minor suggested revisions.\n\nSpeaking of ‘suggestions':\nThe current title ‘Using bio.tools to generate and annotate workbench tool descriptions’ suggests the paper will talk more generally about bio.tools, whereas the text focuses primarily on the specific component ToolDog. The title should be modified to reflect this.\n\nThe graphs in Fig.2 would be more effective if they were displayed in an integrated manner (single bar chart?), so that the improvements that ToolDog makes are more easily compared to one another.\n\nThe discussion about the challenges in autogenerating tool documentation (language, code practices, etc), in the discussion, are spot-on. However not much is discussed on if / how ToolDog might address some of these challenges, though there are suggestions on how to more readily map existing tool descriptions to add to or update. Maybe this could be elaborated on, even if it’s indicating the problems may not be easily overcome?\n\nI’m wondering whether the information in Fig. 5 might be better displayed (or augmented) as a before / after comparison to more readily demonstrate how ToolDog could automatically improve tool descriptions. Another option is whether this information could be somehow connected to the data in Fig. 2 to show how ToolDog improves the overall documentation.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "28567", "date": "10 Jan 2018", "name": "Manuel Corpas", "expertise": [ "Reviewer Expertise Computational Genomics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article 'Using bio.tools to generate and annotate workbench tool descriptions' describes the software tool ToolDog. ToolDog improves the interoperability of bio.tool-deposited entries within workbenches by converting their descriptions into formats that are compatible with workflow standards.\nToolDog is a convenient addition to the existing capabilities for the integration of bio.tools entries with workbench environments.\nI found Figure 2 particularly interesting, describing the metadata coverage descriptions from two of the main Galaxy servers. Do you have the raw data with which this figure was created? It would be good to have it openly shared. Figure 2 illustrates the problem of the significant lack of completeness in crucial metadata descriptions of Galaxy tools.\nMy main recommendation for this article would be to provide a step-by-step guide on how to run ToolDog using a self-contained example. I feel unable to test the tool because I do not know how to download the metadata from a bio.tools entry and need to set up my python environment, download the code and make it work. This article, although it is geared toward a programmer audience, it would be hard to test/reproduce for someone who is not a seasoned python programmer. I would thus recommend a beginner's guide for those of us who are not so technical.\nOther than that, I am glad to see all the source code adequately deposited both in github and Zenodo for the snapshot image for this publication. The MIT license is also commendable as it allows free reuse and modification.\nFinally some minor corrections:\nLink in the first paragraph of the results section ‘of a significant portion of the tool description’ is broken Link on the second paragraph of the results section ‘https://github.com/khillion/galaxyxml-analysis/annotate_usegalaxy’ is broken Discussion section bullet point #3 ‘such web service’ ==> such as web services\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "28595", "date": "16 Jan 2018", "name": "Brian O'Connor", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\n\"Using bio.tools to generate and annotate workbench tool descriptions\" is an article that describes a tool descriptor program known as ToolDog. It was designed to generate Galaxy XML or CWL from particular bioinformatics tool source code as well as metadata annotations on bio.tools. The idea is great, since the issue is a real one in the community. Namely, there are a lot of tools out there but typically they lack descriptors in Galaxy of CWL format. And this makes it harder to use in \"workbench\" and workflow systems. Creating a tool that tool authors can use to help create descriptors is awesome. Source is available in GitHub and the tool can be installed via pip.\n\nFeedback/Questions\nCan the authors rename the article? I think it should include ToolDog in the article title.\n\nWhat are the plans for other languages (if any)? Do the authors see ToolDog as something that others will extend for, say, WDL generation?\n\nI think it would be interesting to hear more about future plans. Specifically, how will the authors expand this to a Workbench Integration Enabler? Do they see this as being an automated process? How will they leverage the work of bioconda and biocontainers (they did mention this briefly) and will the goal be to generate CWL/GalaxyXML for everything in bio.tools + bioconda/biocontainers?\n\nAlternatively, if the goal not to automatically export CWL/GalaxyXML for everything in bio.tools, is it, instead, to provide a tool for tool authors to use when building their tool to jumpstart their descriptor creation? Some clarification on the intended audience I think would be helpful.\n\nThe authors described generating CWL/Galaxy XML for IntegronFinder. Did they try other tools and, if so, how successful was that? What about generation in bulk?\n\nCan they comment on what a tool author should do with the generated CWL or Galaxy XML? They mention in the results that some work is required to make the tool run correctly. Is the tool author then suggested to check in the CWL/Galaxy XML to their source repo and maintain it? What is the recommendation here?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2074
https://f1000research.com/articles/6-1430/v1	11 Aug 17	{ "type": "Review", "title": "Tourette syndrome research highlights from 2016", "authors": [ "Kevin J. Black" ], "abstract": "This article presents highlights chosen from research that appeared during 2016 on Tourette syndrome and other tic disorders. Selected articles felt to represent meaningful advances in the field are briefly summarized.", "keywords": [ "Tourette syndrome", "tic disorders", "review", "animal models", "genetics", "pathophysiology", "therapy", "premonitory" ], "content": "Introduction\n\nThis is the third yearly article in the Tourette syndrome Research Highlights series, intended to share and comment on scientific and clinical advances on Gilles de la Tourette syndrome (TS) and other tic disorders. The highlights from 2017 article is being drafted on the Authorea online authoring platform, and readers are encouraged to add references or give feedback on our selections using the comment feature on that page. After the calendar year ends, the article is submitted as the annual update for the Tics collection on F1000Research.\n\n\nMethods\n\nA PubMed search was conducted using the search strategy “(\"Tic Disorders\" [MeSH] OR Tourette NOT Tourette[AU]) AND 2016[PDAT] NOT 1950:2015[PDAT]”. On 06 Jan 2017, this search returned 186 citations, none of which had first appeared in 2017. The author also identified articles or news from other sources, such as colleagues, email lists, and professional meetings. The studies cited below were selected subjectively, but guided by a personal judgment of their potential importance to the field.\n\n\nResults\n\nEpidemiology. A large, population-based health survey was explored to determine the rate of medically diagnosed TS in Canada1. One in 1,000 respondents had been diagnosed with TS, with the rate higher in youth (0.60%, vs. 0.09% in adults), and in males (risk ratio 5.31). Importantly, those diagnosed with TS had lower total education, income and employment. The results must be interpreted in light of the fact that many people with TS remain undiagnosed, or do not receive medical care.\n\nEnvironmental effects on tic severity. Environmental effects on tics are typical in TS; they are included in the Diagnostic Confidence Index2 and have been demonstrated under careful laboratory conditions3,4. However, typical patient and clinician understanding of TS depends largely on self-report and parental symptom report. Two reports from 2016 highlight some surprising results from prospective observation. First, in one experimental design, tics became less frequent during a short-term psychosocial stressor5. This finding is counterintuitive given the daily experience of patients and observations of clinicians, and supports further research in this area.\n\nBarnea and colleagues recorded video from 41 children age 6–18 with TS in each of five common, daily-life situations6. Their findings were illuminating. First, tic frequency correlated only moderately with reports of children or parents. Self-reported premonitory urges were stronger when patients were more aware of their observed tics. Tic frequency was much higher when children were watching TV and much lower when alone, compared to doing homework, receiving attention when ticcing, or talking to a stranger. The results in the previous sentence differ from typical reports to clinicians, and suggest that developing methods for tic monitoring outside the office may be important to improve reliability and ecological validity in TS research.\n\nTic suppression. Previous studies have attempted to address TS as a generalized failure of action inhibition, but that conceptualization may cast the net too wide7–9. A 2013 publication from Wylie et al.10 had suggested some deficit in response inhibition (action stopping) in TS. In 2016, the group reported a new study in young adults with TS, now better controlled for comorbidity11. Although TS and control groups showed similar speed of cued movement and vocalization, the tic group was slower at stopping these responses.\n\nSensory phenomena. Many patients with TS describe sensory symptoms preceding or independent of their tics. One group recently took a new approach to studying sensation in TS based on a Theory of Event Coding12. Their results suggested that details or features of percepts are less integrated in TS; this finding applied to the group as a whole rather than relating to any obvious symptom or demographic characteristic. The authors speculate as to the possible underlying neurobiology.\n\nPremonitory urges. Premonitory urges are usually reported later in life than tics are first observed. However, a large case series (N>1000) from one clinic suggests that premonitory urges “emerge much earlier than previously thought”: by age 8–10, >60% of children reported premonitory urges, and >75% could suppress tics13. Urges also “were found to be highly associated with ‘not just right experiences’.” The early onset of tic suppression is consistent with a report from the author’s laboratory on tic suppression in the first few months of tic disorder onset14.\n\nBrandt et al.15 performed a careful experiment to investigate the timing of urges in relation to tics, compulsions and—as a comparison to a naturally arising urge—blinks when attempting to keep the eyes open. Another group examined tics and urge to tic at 10- to 15-second intervals in 12 patients with moderate to severe TS; different patients had quite different relationships between urge and tic timing when examined at this temporal scale16. These observations show that careful phenomenological studies still have much to teach us about tic disorders.\n\nOther. The frequency of anxiety and impulsivity in TS has suggested the possibility of a deficit in emotional self-regulation in some patients. A recent study examined specific emotional regulation approaches taken by adults with TS17. The TS and control groups did not differ on anxiety or depression symptom scores, but the TS group used suppression as a strategy more often than the control group.\n\nSelf-injurious behavior (SIB) is an important clinical problem in the minority of TS patients who experience it18. Sambrani et al.13 felt that their data supported lumping SIB with coprophenomena, and argue for it to “be conceptualized as a complex tic rather than a compulsion.” Others have found similar results19, though other results support a connection to compulsions20, one large study found SIB to fit better with ADHD symptoms21, and another linked it to both OCD and ADHD22.\n\nSelf-reported depressive symptoms were as common and severe in TS as in tic-free patients with major depression, though people with TS endorsed irritability more frequently23.\n\nGenetics. An important, collaborative study showed that rare copy number variants (CNVs) increase the risk for TS24,25. This was true both generally for large CNVs, exceeding 1 million base pairs, and for CNVs previously linked to pathology. Specifically, NRXN1 and CNTN6 each conferred a substantially increased risk of TS: 20-fold and 10-fold, respectively. These associations give important impetus to research focusing on the cellular effects of these genes and perhaps related treatments. This research also confirms that the clear genetic risk for TS is spread across numerous genes, because only 1% of patients with TS carry risk alleles in these two genes.\n\nA population-based adult twin study of tics, ADHD and OCD found that the co-occurrence of tics and OCD can be partly explained by shared etiological influences26. The same is true to some extent for tics and ADHD. Another report concluded that social disinhibition in TS is heritable27. More research examining sub-dimensions of these phenotypes may clarify the situation.\n\nAnother potentially useful approach to TS genetic research was illustrated by 28, who sequenced candidate genes suggested by previous research in order to identify potential functional variants linked to TS.\n\nEnvironmental risk factors and epidemiology. Identifying specific environmental risk factors for TS is important in its own right, and also because controlling for known environmental risks can improve the power of genetic studies. Previous studies have linked TS with lower birthweight, and more recently with maternal smoking during gestation29–31. Leivonen et al. reported the first nationwide pseudo-prospective (i.e., based on a Finnish national health registry) study of prenatal maternal smoking and TS32. In this sample, the hypothesized association of maternal smoking with TS per se was not significant, but maternal smoking during pregnancy was significantly associated with combined TS and ADHD, and the odds ratio for TS+ADHD following first trimester smoking was 4.0 (95% CI 1.2–13.5, p=.027). This association provides only modest support for the link between prenatal toxicity and TS. However, this study is consistent with the idea shared by many tic experts that the current nosology artificially separates tics from other common symptoms in people with TS.\n\nAnimal models. A face-valid animal model of tics has been a very important but elusive goal33. McCairn and colleagues have now published their important work characterizing a nonhuman primate tic model34. Temporary unilateral disinhibition of the nucleus accumbens (NA)—the ventral, more limbic-connected part of the striatum—produces vocalizations in monkeys that resemble vocal tics in human patients. The vocalizations sometimes were associated with local field potential (LFP) spikes in the NA, but not always; in the absence of local LFP spikes, phase coupling in the alpha frequency band between NA, anterior cingulate and primary motor cortex tended to be stronger. By contrast, disinhibition of the (more traditionally motor) dorsolateral putamen led to repetitive movements that the authors call “myoclonic tics,” whose phenomenology is less similar on its face to that of tics, and which were regularly preceded by LFP spikes.\n\nVasoactive intestinal peptide-expressing interneurons were targeted for developmental injury using a conditional expression model in mice35. These mice showed hyperactivity and altered firing of some cells at the onset of the transition from quiescence to locomotion. How this may relate to lower numbers of parvalbumin-containing interneurons in TS36 is unclear. An interesting contrast based on the parvalbumin data is provided by a model in which fast-spiking interneurons in the dorsal striatum were ablated37. These mice developed anxiety and increased frequency of grooming rituals.\n\nIn another rodent model, D1CT-7 mice—in which dopamine D1 receptors are targeted— were compared to wild-type mice38. Spatial confinement triggered stress reflected by corticosterone release in both groups, but exacerbated abnormal behaviors in the D1CT-7 mice, such as digging, biting, jumping and motor perseveration. Stress also led to impaired prepulse inhibition, thought of as reflecting sensorimotor gating. Both the abnormal motor behavior and the prepulse inhibition deficit improved with clonidine, haloperidol, or SCH23390 (a dopamine D1 receptor antagonist). Collectively, these results support the potential utility of this model in screening new treatments for tic disorders.\n\nFinally, spontaneously-occurring models of tics in other animals are of great interest39. Kalueff and colleagues provide a helpful review of the substantial body of knowledge available about stereotyped grooming patterns in rodents40, arguing appropriately that these patterns may be “useful for understanding the neural circuits that are involved in complex sequential patterns of action” in human disease, including in TS41.\n\nElectrophysiology. In contrast to findings from previous studies in older patients, resting motor threshold and variability of motor evoked potential responses in 17 TS subjects, age 12–22, were increased versus controls, while the gain of motor excitability was reduced42. The authors suggest that these findings may relate to delayed maturation of the cortical-cortical and corticospinal motor networks in TS.\n\nLocal field potentials (LFP) in the posterolateral globus pallidus pars interna (GPi) were recorded at rest and during tics and normal voluntary movements in three adults with TS during deep brain stimulation surgery and compared to GPi LFP recordings in four Parkinson disease patients off medication43. Tics were associated with increased gamma (35–200 Hz) and high frequency (200–400 Hz) activity, and with cross-frequency coupling (between the phase of beta band activity and the amplitude of the high-frequency oscillations).\n\nNegative reinforcement learning is a key variable in theories of behavior therapies proven effective for TS. In this context, the findings of a 2016 report from Nottingham, England, are interesting. Behavioral and event-related potential data were collected during the learning of stimulus-response pairs using positive or negative reinforcement44. Participants included children age 9–17 with TS, ADHD, both or neither. Lower accuracy and smaller P3 amplitudes were observed in association with ADHD, but not TS.\n\nO’Connor and colleagues have developed a cognitive-behavioral therapy approach to tic treatment, discussed below in the Treatment section, based in part on electrophysiological findings. For instance, previous studies had identified a reduced P300 oddball effect in TS patients with or without OCD. This group has now reported a study of event-related potentials in adults with TS or a body-focused repetitive behavior disorder (such as trichotillomania) in comparison to a healthy control group45. The P300 oddball effect was lower in both patient groups before treatment, and increased with cognitive-psychophysiological treatment.\n\nNeuroimaging studies. A structural brain MRI study in 103 children with TS, age 7–17 years, and 103 matched control subjects, found greater gray matter volume in TS in the posterior thalamus, hypothalamus and midbrain, and decreased white matter volume in orbital prefrontal and anterior cingulate cortex46. Structural abnormalities in these regions was hypothesized to influence somatosensory processing, decision making or reinforcement learning. Eddy and colleagues continued their research using theory of mind (ToM) approaches to TS, reporting on an fMRI study involving a task that required the subject to infer another person’s thoughts when making decisions47. TS subjects showed overall lower responses to the ToM task and to a control task, but also in several regions a lower increase with the ToM task compared to the control task. These regions included the posterior cingulate, right angular gyrus and right amygdala, regions involved in previous ToM studies in people without tics. Group differences correlated with various symptom severity measures, including a strong correlation of task-related temporoparietal junction activation with premonitory urge severity.\n\nSPECT imaging with a perfusion marker was used to investigate the possible mechanism of action of deep brain stimulation in the internal GPi and in the medial thalamus48. Controls included sham stimulation in the TS group and unstimulated subjects without tics. Active stimulation in either site decreased regional blood flow in basal ganglia and cerebellum and increased perfusion to the frontal cortex, including the supplementary motor area.\n\nBrain imaging studies include very large data sets, and have required development of novel statistical methods. Lessov-Schlaggar et al. argued thoughtfully that typical statistical analyses of a single variable at a time may be inadequate to find many important solutions to “complex disorders of brain development and function”, such as TS49.\n\nPharmacological studies. German researchers reported a fascinating study in 37 medication-free adults with TS and 36 controls using magnetic resonance spectroscopy (MRS)50. The study tested the hypothesis that GABA, glutamine (Gln) and glutamate (Glu) concentrations in striatum and thalamus would differ in people with TS. Glutamine, Gln+Glu and the Gln:Glu ratio were lower in TS, more so in the striatum than in the thalamus. The striatal Gln concentration correlated negatively with tic severity on the day of the scan, and thalamic Glu concentration correlated negatively with premonitory urge severity. Surprisingly, these abnormalities tended to improve during treatment with aripiprazole. The authors work hard to minimize any effects of head motion, which might otherwise confound these results. The results overall suggest an abnormality in glutamatergic transmission in TS. Readers who are not MRS gurus may benefit from examining their Supplementary Figure 5, which demonstrates the challenges in eking out signal for these compounds.\n\nGremel et al.51 present data from an optogenetic study in mice, showing that removing cannabinoid type 1 (CB1) receptors from orbitofrontal cortex efferents to dorsal striatum prevents the mice from graduating from intentional to habitual lever pressing. The authors conclude that this cannabinoid-modulated orbitofrontal-striatal pathway is essential to the development of habits. As tics may be conceptualized as involving overenthusiastic habit formation circuitry, one might have expected the opposite result, given anecdotal beneficial short-term effects of cannabinoids on tic severity52.\n\nIn another mouse model, knocking out the histamine H3 receptor impaired prepulse inhibition, and abolished or reduced dopaminergic signaling via both D1 and D2 receptors53. These results, combined with previous studies linking histamine to TS, support the development of histamine H3 receptor ligands for possible use in TS.\n\nClinical and neuropsychological studies. Thresholds for externally applied sensory stimuli were similar in adults with “pure” TS and controls54. These results, like those of a previous study55, demonstrate that the sensory symptoms of TS are central in origin, suggesting abnormalities in interoceptive awareness or central sensorimotor processing.\n\nOne interesting study focused on the clue that classic features of TS include echopraxia and suggestibility (the triggering of a tic by discussing or seeing it). Following up on their previous work56,57, Münchau and colleagues asked tic-free human control participants to imitate separately a tic and another, non-tic facial movement from a matched participant with TS58. While doing so, subjects watched video of one of the two movements. For non-tic movements, TS subjects responded like controls, in that movements were performed more quickly when the video was congruent with the requested movement than when it was incongruent. However, when asked to execute a tic, TS subjects responded equally quickly regardless of the video content. The authors conclude that “tic-like movements do not occur as a consequence of a failure to inhibit motor output. Instead, tics might be considered highly overlearned behavior that can be triggered without interference by external, incompatible movement stimuli.”\n\nMost adults with TS feel that at least some of their tics are an eventual voluntary capitulation to an almost irresistible urge, though some tics are felt to be more truly involuntary. Researchers from Paris reported an interesting study of how adults with TS judge agency (the sense that one initiates and causes one’s own actions)59. The study involved manipulating in several different ways whether a participant’s hand movements were accurately reflected in the movements of a computer cursor. Those with TS recognized when their intended movements were substantially altered, yet were less aware that their intentions had been altered when the alterations comprised enhancement of their performance. These results add to a prior study of agency in adolescents with TS, in which awareness of their intention to move was earlier in those with greater tic suppression ability and later in those with more severe premonitory urges60. Together, these studies suggest that the sense of agency with intentional movement differs subtly in people with TS, and may relate to patients’ judgments of whether their tics are volitional.\n\nA meta-analysis of tic treatment discusses patient satisfaction in addition to efficacy and side effects, and concludes that there is good evidence supporting a favorable benefits:harms ratio for the most commonly used medications, but that “larger and better-conducted trials addressing important clinical uncertainties are [still] required”61.\n\nPsychological interventions. Behavior therapy is now an accepted first-line treatment for tic disorders. Still, some practitioners retain misconceptions about its safety. A report on 228 participants in randomized controlled trials (RCTs) of Comprehensive Behavioral Intervention for Tics (CBIT) helps to confute those concerns62. Specifically, CBIT participants were no more likely to have new tics, have adverse events, increase tic medications or have an exacerbation in psychiatric symptoms relative to patients who received supportive therapy.\n\nFactors that slow wider implementation of behavior therapies for tics include cost and a limited number of trained practitioners. Group (rather than individual) therapy is one option that could address these obstacles. A small study provided initial testing of this possibility using a treatment and a control group. Both groups had improvements in quality of life, and the greatest improvements in motor tic severity occurred in the HRT group63. The most important conclusion from this pilot study was that the group format was acceptable and appears not to impair efficacy.\n\nAnother potential solution to the implementation concerns noted above is providing therapy online. Müller-Vahl and colleagues are beginning a large RCT that will test the efficacy of CBIT delivered by internet64. A commercial CBIT delivery system has been described, but (as of mid-2017) is not yet active.\n\nResearch continues on other psychotherapy approaches. Suppressing tics is generally uncomfortable—leading to the theory of negative reinforcement that motivates CBIT and exposure and response prevention approaches to tic treatment—and in recent years Acceptance and Commitment Therapy for various conditions has become more popular, and pilot studies have appeared applying this approach to tics65,66. In this setting, Gev et al.67 report their study of 45 children and adolescents with TS who rated severity of tic urges, and were observed, during each of three 2-minute conditions: baseline (ticcing freely), tic suppression, and urge acceptance. Urges and discomfort decreased significantly during the acceptance condition, and more importantly, so did tic frequency. By contrast, tic urge intensity and self-rated discomfort increased during the tic suppression condition. These findings suggest that acceptance therapy may be better—accepted—by patients. It is now ripe for tests of long-term efficacy.\n\nFor some years, O’Connor, Leclerc and colleagues have developed and utilized a cognitive-behavioral-psychophysical treatment model for tic disorders. The treatment aims to address sensorimotor function more broadly, including proprioception, response inhibition and perfectionism, rather than focusing on tics directly. In 2016, several important publications from their group addressed this approach, including an open study of 102 adults with a chronic tic disorder68, in which tic severity improved for both simple and complex tics, and the improvement remained at 6 months. Measures of self-esteem and perfectionism also improved. A pilot study in children age 8–16 was also reported69, followed by initial results with a manualized version of this therapy in children age 8–1270. This “cognitive-psychophysiological treatment” model provides another potential approach to treatment that may prove to have more generalizable benefits.\n\nMedication. An open-label, rater-blind, 8-week study of deutetrabenazine in TS provided positive results71. This compound is a deuterium-substituted tetrabenazine (a VMAT2 inhibitor) with slower elimination half-life, and was approved by the U.S. Food and Drug Administration (FDA) in 2017 as a treatment for chorea in Huntington disease, making it potentially available for off-label use for tics. Similarly, another VMAT 2 inhibitor, valbenazine, was approved in 2017 for treatment of tardive dyskinesia.\n\nAdding to prior evidence of anti-tic efficacy, a prospective, open case series in 44 adults with TS found that aripiprazole helped non-tic symptoms as well as tics, though premonitory urges to tic were not significantly improved72. However, in May 2016, the FDA warned that aripiprazole can cause disinhibition in the form of impulse control disorders73. Similar side effects have been described for levodopa and dopamine agonists, but had not been recognized for aripiprazole, which is a partial agonist at dopamine receptors.\n\nOther medication trials reported in 2016 include a failed add-on RCT with N-acetyl-cysteine74 and a case series suggesting iron supplementation may improve tics in TS patients with low serum ferritin75.\n\nNeurosurgery. Surgery is an option for carefully selected patients with TS, yet several important questions remain76. The Galeazzi Institute in Milan, Italy, provided a progress report on their large sample of TS patients treated with deep brain stimulation (DBS), mostly in the ventromedial thalamus77. In 11 of 48 patients (23%), the device was removed after “inflammatory complications” or poor compliance with follow-up. In the remaining 37 patients, 29 had a more than 50% reduction in YGTSS scores (clinician-rated tic severity and impairment). In a separate publication, they argued that the patient’s symptoms beyond tics should be considered when a DBS target is selected78. Hartmann argued contrariwise that our current state of knowledge better supports a narrower focus on tic reduction in choosing a DBS target79.\n\nThe TS group from Maastricht, The Netherlands, reported positive unblinded follow-up results in five patients with refractory TS treated with DBS in the anterior GPi80, and a Chinese group reported unblinded 1-year follow-up of GPi DBS in 24 patients with TS, with improvement, on average, in both tics and OCD symptoms (50% reduction in mean YGTSS total tic score and 27% reduction in mean Y-BOCS score; Supplementary File 1)81. Interestingly, this latter report includes one patient whose tics continued to improve after the DBS electrode was removed (p. 1025), consistent either with spontaneous improvement over time or with a micropallidotomy lesion effect.\n\nDBS in a mouse model suggests that self-injurious behaviors, probably over-represented in TS patients referred for surgery, may be amenable to DBS of the subthalamic nucleus82.\n\nGiven the current lack of consensus on DBS methods in TS, gathering data on all DBS patient outcomes in TS is crucial, and a recent collaborative report described the establishment of the International Deep Brain Stimulation Registry and Database for Gilles de la Tourette syndrome83.\n\nOther treatment. Many patients have reported to their doctor that music performance reduces their tic symptoms. Brown84 addressed this formally, surveying 183 musicians diagnosed with TS. On a scale of 1 (drastic symptom worsening) to 5 (drastic symptom improvement), subjects reported a mean of 4.45 for the effect of engaging in a musical activity (performance, not passive listening). This result strongly supports the patient anecdotes and suggests that formal music therapy could be tested in a randomized controlled trial for benefit on TS symptoms.\n\nSome tic patients have been very interested in PANDAS, a name representing the hypothesis that obsessions, compulsions and tics represent an aberrant immune response to streptococcal infection in some children85. Recently a second double-blind study of intravenous immunoglobulin (IVIG) for OCD symptoms in PANDAS patients was published86. Unfortunately, although effects during the open label phase were substantial, there was little difference between the IVIG and control groups during the double-blind portion of the study.\n\nThree tic experts contributed a thoughtful review of the effect of tics and non-tic symptoms (or comorbidities, depending upon one’s nosological viewpoint) on social relationships and quality of life in TS87. Wadman and colleagues performed an in-depth study of 35 youth with TS, their parents, and school personnel to understand what problems TS caused at school88. Their conclusion will seem sadly familiar to TS patients and clinicians: “Young people and parents agreed more strongly with each other than they did with staff regarding school difficulties faced by individuals, and staff generally reported fewer TS-related difficulties.”\n\nThe first Frontiers Research Topic on TS was published this year, including over 30 articles on TS, many stemming from the 2015 conference in London89. Thenganatt and Jankovic provided a useful review of TS90. A comprehensive review of Provisional Tic Disorder also appeared91; this diagnosis refers to children whose first tic was less than a year ago, similar to Transient Tic Disorder in earlier nosologies. A helpful overview of TS genetics92, and a review of epigenetic mechanisms and how they may prove to affect TS, both appeared in mid-201693.\n\n\nConclusions\n\nAdvances in many areas of TS research and clinical care were reported in 2016. Smaller pilot studies remain important for purposes other than settling a question: they can be useful for testing feasibility, for improving methods, and for identifying new hypotheses. (In fact, one of the most significant publications in TS history was a single-blind N-of-1 study94.) Several of the studies mentioned above are small studies that fulfill one or more of these goals.\n\nNevertheless, as the title of the 2016 Frontiers Research Topic suggests, TS research is beginning to grow from case series and small pilot studies into finding “new avenues through large-scale collaborative projects.” This change is important, especially in light of increasing recognition that reproducibility is an important concern in all of science, and clearly in neuroscience95,96. Larger samples are important to addressing this concern, and give greater hope for important new discoveries in the future.", "appendix": "Competing interests\n\n\n\nKJB participated in clinical trials supported by Psyadon Pharmaceuticals and Neurocrine Biosciences, Inc.\n\n\nGrant information\n\nThis work was supported in part by the U.S. National Institutes of Health (NIH) (grants R21 NS091635 and R01 MH104030), and by a research grant from the Tourette Association of America (PI: Cheryl A. Richards, Ph.D.).\n\nThe author confirms that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplemental material\n\nSupplementary File 1: Calculation of the percent improvement numbers from the Zhang et al DBS report81.\n\nClick here to access the data.\n\n\nReferences\n\nYang J, Hirsch L, Martino D, et al.: The prevalence of diagnosed Tourette syndrome in Canada: A national population-based study. Mov Disord. 2016; 31(11): 1658–1663. PubMed Abstract \| Publisher Full Text\n\nRobertson MM, Banerjee S, Kurlan R, et al.: The Tourette syndrome diagnostic confidence index: development and clinical associations. Neurology. 1999; 53(9): 2108–12. PubMed Abstract \| Publisher Full Text\n\nGoetz CG, Leurgans S, Chmura TA: Home alone: methods to maximize tic expression for objective videotape assessments in Gilles de la Tourette syndrome. Mov Disord. 2001; 16(4): 693–7. 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Coffman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript submitted by Dr. Black is a very thorough and comprehensive review of the recent literature on Tourette Syndrome. It is exceptionally well written and organized.\n\nThere is one error that needs to be corrected. In the following sentence, there is a missing word and simply a citation number.\n\"Another potentially useful approach to TS genetic research was illustrated by 28, who sequenced candidate genes suggested by previous research in order to identify potential functional variants linked to TS.\"\nOther than this, I could find no errors or modifications that I would recommend before publishing this manuscript.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "2986", "date": "29 Aug 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "Thank you! I can correct the missing author name in a revision after other reviews are back." } ] }, { "id": "25610", "date": "12 Sep 2017", "name": "James F. Leckman", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nProf. Kevin Black has provided an up-to-date and largely comprehensive summary of the current status of our knowledge concerning the phenomenology, natural history, etiology, pathophysiology, and treatment of Tourette syndrome (TS). However, one important area that is largely missing concerns the findings from post-mortem studies of the brains of individuals with TS that have been conducted by Flora Vaccarino and her laboratory at Yale University. The most notable and highly-cited article from 2016 presents an analysis of the transcriptome of striatal tissue (caudate and putamen) from the brains of nine TS and nine matched normal control subjects (Lennington et al., 20161). The authors found 309 down-regulated and 822 up-regulated genes using a data-driven gene network analysis. More specifically, they identified 17 gene co-expression modules associated with TS. The top-scoring down-regulated module in TS was enriched for striatal interneuron transcripts. This finding confirmed earlier studies by the Vaccarino laboratory that had reported decreased numbers of cholinergic and gamma-aminobutyric acidergic interneurons in the same brain regions (Kalathini et al., 2005; Kataoka et al., 20102). However, the top-scoring up-regulated module was enriched in immune-related genes, consistent with activation of microglia in patients’ striatum. While these findings confirm the earlier post-mortem studies using unbiased stereological techniques, they also point to the important role of neuroinflammation in TS pathophysiology. Indeed, a deeper understanding of neuroimmunology may well transform the field of neuropsychiatry and point to novel treatment approaches to TS and related disorders (Leckman & Vaccarino, 20153).\n\nIs the topic of the review discussed comprehensively in the context of the current literature? No\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3020", "date": "13 Sep 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "I agree with the importance of the Lennington et al., 2016, paper.1 It is absent from the \"from 2016\" highlights article only because (since the final version appeared online before its official publication date) we discussed it in the 2015 highlights article2 (first paragraph under Pathophysiology). We cited the Kataoka et al publication3 in the 2014 highlights paper.4 I do appreciate the reference to the 2015 editorial,5 which together with your review above better highlights the relevance of the Lennington et al. report to the broader question of neuroimmunology in Tourette syndrome. Lennington JB, Coppola G, Kataoka-Sasaki Y, Fernandez TV, Palejev D, Li Y, Huttner A, Pletikos M, Sestan N, Leckman JF, Vaccarino FM: Transcriptome Analysis of the Human Striatum in Tourette Syndrome.Biol Psychiatry. 2016; 79 (5): 372-82 PubMed Abstract \| Publisher Full Text Richards CA and Black KJ. Tourette syndrome research highlights 2015 [version 1; referees: 3 approved]. F1000Research 2016, 5:1493 (doi: 10.12688/f1000research.8769.1) Kataoka Y, Kalanithi PS, Grantz H, Schwartz ML, Saper C, Leckman JF, Vaccarino FM: Decreased number of parvalbumin and cholinergic interneurons in the striatum of individuals with Tourette syndrome.J Comp Neurol. 2010; 518 (3): 277-91 PubMed Abstract \| Publisher Full Text Richards CA and Black KJ. Tourette Syndrome research highlights 2014 [version 2; referees: 1 approved, 2 approved with reservations]. F1000Research 2015, 4:69 (doi: 10.12688/f1000research.6209.2) Leckman JF, Vaccarino FM: Editorial commentary:. Brain Res. 2015; 1617: 1-6 PubMed Abstract \| Publisher Full Text" } ] }, { "id": "25611", "date": "28 Sep 2017", "name": "Davide Martino", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors present an interesting and comprehensive review of highlighted articles published in 2016 on Tourette syndrome. The article is very well written.\nThere are a few articles that might also be considered for highlight, in addition to those already referenced.\nComorbidities:\nJohnco C et al. (2016)1 This is particularly relevant in view also of an important finding coming from a 2017 population-based study (Fernandez de la Cruz, Biol Psychiatry 2017)\nGenetics:\nBertelsen B et al. (2016)2\nEnvironmental factors (etiology):\nAbdulkadir M et al. (2016)3\nImaging:\nWen H et al. (2016)4 Liao W et al. (2017)5\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3068", "date": "29 Sep 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "Thanks for your input. These are excellent suggestions and I plan to include them in a revision." }, { "c_id": "3198", "date": "29 Nov 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "We cited the Bertelsen et al paper in our 2015 highlights paper [1]. The Liao et al paper [2] uses a relatively lenient threshold for censoring head motion [3], complicating interpretation. I've included your other suggestions in version 2.[1] Richards CA and Black KJ. Tourette syndrome research highlights 2015 [version 1; referees: 3 approved]. F1000Research 2016, 5:1493 (doi: 10.12688/f1000research.8769.1).[2] Liao W, Yu Y, Miao HH, Feng YX, Ji GJ, Feng JH: Inter-hemispheric Intrinsic Connectivity as a Neuromarker for the Diagnosis of Boys with Tourette Syndrome.Mol Neurobiol. 2017; 54 (4): 2781-2789 PubMed Abstract \| Publisher Full Text[3] Power JD, Mitra A, Laumann TO, Snyder AZ, Schlaggar BL, Petersen SE: Methods to detect, characterize, and remove motion artifact in resting state fMRI. Neuroimage 2014; 84:320-341 (doi: 10.1016/j.neuroimage.2013.08.048)" } ] }, { "id": "25613", "date": "04 Oct 2017", "name": "Christos Ganos", "expertise": [ "Reviewer Expertise Tourette Syndrome", "Dystonia and other Movement Disorders" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI thoroughly enjoyed the \"Tourette Syndrome research highlights from 2016\" article by Prof. Black.\nIt is a very well organised effort to present a succinct summary of relevant research from that year and I would like to congratulate Prof. Black for his dedication in putting this together.\n\nI only have some minor suggestions.\nIt might be thematically more suitable to integrate the first paragraph of the sensory findings of the \"Clinical and neuropsychological studies\" [\"Thresholds for externally applied sensory... ...or central sensorimotor processing\"] to the \"Premonitory Urges\" section, as indeed these refer to the same topic.\n\nOn the reference of the mouse model of Xu et al. [\"These mice developed anxiety and increased frequency of grooming rituals.\"] it might be of relevance to highlight that the abnormal behaviours only occurred after exposure to environmental stressors.\n\nOn the Treatments/psychological interventions section, it could be relevant to mention that CBIT responders in those trials as measured by the CGI-I were 45% of the total sample. [Data from the cited publication: \"CBIT were significantly more likely to be classified as treatment responders on the CGI-I (child = 53%, adult = 38%, combined sample = 45%)\"]\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3085", "date": "05 Oct 2017", "name": "Kevin J Black", "role": "Author Response F1000Research Advisory Board Member", "response": "Thanks for the kind comments and expert suggestions, which I can include in a revision." } ] } ]	1	https://f1000research.com/articles/6-1430
https://f1000research.com/articles/6-2068/v1	29 Nov 17	{ "type": "Research Article", "title": "Polypharmacy in Alzheimer's disease patients in Brazil: Guidance for pharmaceutical assistance", "authors": [ "Felipe Nathanael Coelho Vaz", "Luana Bortoluzzi Trombim", "Guilherme Barroso L. de Freitas", "Maria Vaitsa Loch Haskel", "Giovana dos Santos", "Jéssica Wouk", "Dayanna Hartmann Cambruzzi Mendes", "Barbara Luisa Fermino", "Flávia Ivanski", "Juliana Sartori Bonini", "Felipe Nathanael Coelho Vaz", "Luana Bortoluzzi Trombim", "Guilherme Barroso L. de Freitas", "Maria Vaitsa Loch Haskel", "Giovana dos Santos", "Jéssica Wouk", "Dayanna Hartmann Cambruzzi Mendes", "Barbara Luisa Fermino", "Flávia Ivanski" ], "abstract": "Background: Elderly patients frequently have concomitant diseases, triggering the necessity of utilizing several different medications, which can cause adverse events associated with therapy, called polypharmacy. This study aimed to evaluate the main concomitant diseases with Alzheimer's disease (AD) and discuss possible interactions between drugs utilized to treat dementia and its comorbidities, and indicate safe medicines for patients with AD. Methods: 41 individuals with AD who withdraw medicines for dementia from the Brazilian public health system (SUS) participated in this study. Data collection was performed using three questionnaires: 1) Clinical Dementia Rating, to verify disease stage; 2) Mini–mental state examination, to measure cognitive impairment; and 3) Sociodemographic analysis, to evaluate concomitant diseases, utilized drugs, drug-drug interactions, among other demographic variables. Statistical analyses were performed using SPSS and data was presented as relative frequency. Results: The results of this study showed that the most frequent concomitant diseases with AD are: systemic arterial hypertension, depression, diabetes mellitus, and hypercholesterolemia. Polypharmacy was observed in 95.12% of patients. The pharmacologic classes that presented interactions with AD medications were anxiolytics, antidepressants, antipsychotics, antihypertensives, and antidiabetics. Conclusion: In the present study, polypharmacy in patients with AD and other concomitant diseases has been characterized. The average number of drugs that these patients ingested was seven per day, and this leads to drug interactions, which are potentially damaging to the body. Consequently, we have tried to reduce these interactions, by suggesting drugs that are safer, for example furosemide instead of amlodipine to treat hypertension.", "keywords": [ "Medicine usage", "Elderly", "Pharmacoepidemiology", "Alzheimer’s Disease" ], "content": "Introduction\n\nAlzheimer’s disease (AD) is characterized by beta-amyloid (βA) peptide production and aggregation in specific regions of the brain, such as the hippocampus, and ventral and entorhinal cortex1. AD is the most common dementia, marked by progressive cognitive and motor impairments. This disease compromises patients’ daily life activities2, affecting attention, language, visual-spatial ability, locomotion and primarily, memory3.\n\nElderly patients are at considerably higher risk of developing conditions such as cancer, diabetes, inflammations, and cardiovascular and neurodegenerative diseases, e.g., AD and Parkinson’s disease4. Therefore, elderly patients frequently have concomitant diseases, triggering the necessity of utilizing several different medications.\n\nPharmacology is distinct in elderly patients because, during the process of aging, some alterations are observed in body composition and renal and hepatic functions, interfering in the pharmacokinetics and pharmacodynamics of several drugs; for these reasons, elderly patients are more vulnerable to iatrogenesis4.\n\nAdverse events caused by the concomitant use of several drugs may be prevented by making an adequate prescription. Potential inappropriate medications (PIMs) are drugs with a high-risk of provoking more side effects than benefits, even though there are available alternatives that can be substituted5. In Brazil, PIMs are still being prescribed and used as top-notch treatments for the majority of elderly patients, although there is evidence of negative results6,7. This occurs because these medications are in the Brazilian National Essential Medicines List (RENAME) and are distributed free of charge by the Brazilian public health system (SUS)5.\n\nAmong elderly patients, adverse events associated with medications are caused by polypharmacy, which facilitates adverse drug reaction (ADR) and drug interactions8. According to Ribeiro and colleagues (2013)9, polypharmacy may be classified as mild, moderate and grave, depending on if the patients utilize 2-3, 4-5, or 5+ medications, respectively10,11.\n\nIndividualized healthcare is essential for elderly patients with polypharmacy. Therefore, protocols have been developed that aim to establish appropriate drug prescription for elderly patients. The most employed protocols are the PRISCUS list12 and Beers-Fick criteria13. PRISCUS list is more updated and inclusive; however, both protocols are not complete or adapted to Brazilian ambulatory reality. For that reason, the present study aimed to verify the most frequent diseases concomitant with AD and analyze the interactions between medications and these diseases, to indicate a safer alternative treatment for AD patients.\n\n\nMethods\n\nThis research was approved by the Ethics Committee of Research of Midwest State University (COMEP/ UNICENTRO; Guarapuava, Brazil), approval no. 968931.\n\nThis study was conducted between March 2015 and July 2016. Elderly patients invited to participate in this study had a confirmed AD diagnosis (inclusion criteria), issued by a geriatric or neurologist, according to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer Disease and Related Disorders Association (NINCDS- ADRDA) criteria14. All participants received free medicines from SUS for disease treatment. Those without confirmed diagnosis, that were absent after three consecutive home visits, that changed residence or died before blood collection were excluded from the study.\n\n57 elderly patients with AD were randomly invited to participate in this study, but only 41 reached the end of the study. Initially, phone calls were made by the researchers to explain the objective and purpose of the research, who were recruited at Basic Health Unit (UBS) of Vila Carli, Industrial, Santana, Santa Cruz e Paz e Bem. All are characterized as low level health facilities in Guarapuava/PR city. If the participant accepted the invitation, a meeting was scheduled (home visit) with the caregivers to present and sign the informed consent form (if patients were lucid, they signed a consent form, but, if not, the caregivers provided the written consent). Subsequently, by an interviewer, three questionnaires were applied to the AD patients: Clinical Dementia Rating (CDR), Mini-Mental State Examination (MMSE) and a sociodemographic questionnaire.\n\nClinical Dementia Rating (CDR)14 aims to classify the disease’s stage in CDR-1, 2 or 3, which indicates mild, moderate and severe dementia, respectively. In contrast, Mini-Mental State Examination (MMSE)15 evaluates global cognitive functions and was applied as a psychometric analysis of orientation, attention, calculation, and language. The maximum score for MMSE is 30, and this indicates cognitive impairment. The sociodemographic questionnaire (Supplementary File 1) analyzed the patient’s profile, knowledge about their diseases and identifying drugs and dosages utilized daily.\n\nAfter discussing patients’ characteristics, a drug-interaction analysis was performed by Scientific studies, Beers-Fick and PRISCUS protocols, and medical studies were analyzed to verify drug-drug interactions, in addition to using the drugs.com database, in which, for each patient, a folder was created and inserted all medicines. At the end of the process the drugs.com base returned a report with the interactions. Each medication received a code: 0 to the absence of interaction and 1 to the presence of any interaction with an AD medication (Supplementary File 2).\n\nStatistical analyses were performed using SPSS® software version 20.0, utilizing operational system Windows 10 Pro® and Office 2016® package. The results were presented in relative and absolute frequency.\n\n\nResults\n\nFrom the initial sample of 57 individuals, eight (14.04%) died before data collection, and eight (14.04%) were absent after three consecutive home visits. The final sample total 41 patients.\n\nResults of CDR test showed most of the patients were in CDR 3, AD severe stage (Table 1). Because of that, patients presented with higher cognitive impairment, and consequently, the proposed questionnaires could not be responded to properly. Therefore, the mean number of correct answers in MMSE was 10.80. Regarding concomitant diseases, systemic arterial hypertension was the most frequent (58.54%), followed by depression (46.34%), diabetes mellitus (27.28%), and hypercholesterolemia (26.80%).\n\nData are presented as mean ± standard deviation; relative frequency. CDR: Clinical Dementia Rating14; MMSE: Mini-Mental State Examination15.\n\n63.42% (n=26) of the patients took AD medications that interacted with drugs taken to treat other diseases (Table 2). Drug-interactions occurred more frequently in patients with the moderate stage of AD (CDR-2, 68.76%), followed by the mild stage (CDR-1, 66.67%) and lastly, patients with severe stage AD (CDR-3, 57.89%).\n\nData were presented in relative frequency.\n\nAccording to the results shown in Table 3, 34 of 41 elderly patients, took AD medications. Of these, half (50%; n=17) utilized Donepezil hydrochloride and 38.24% (n=13) utilized rivastigmine, both acetylcholinesterase inhibitors (AChEI). Only four patients (11.76%) utilized memantine, an adjuvant drug to AD treatment, which blocks N-Methyl-D-aspartate receptor (NMDAR), decreasing mitochondrial oxidative stress. Memantine must only be used during mild and moderate stages of AD (CDR-1 and 2). Thus, two patients diagnosed with severe AD (CDR-3), were ineffectively treated with memantine.\n\nData presented as relative frequency.\n\nFrom 19 patients with CDR 3, 36.84% (n=7) did not use any AD-specific drug, due to Brazilian legislation (Ordinance SAS/MS No. 1.298 of November 21, 2013). This legislation does not allow patients in AD severe stage (CDR 3) to withdraw medications from SUS, claiming a low efficiency of AChEI treatment. Medications utilized to treat concomitant diseases are fully described in Table 4.\n\nSource: GOODMAN & GILMAN16, 2012; Sociodemographic questionnaire.\n\nAD treatment consists of AChEI (rivastigmine and donepezil) and NMDAR antagonists (memantine). Therefore, knowing which medications interact with these drugs is fundamental to indicate the correct treatment for secondary diseases and, even, predict drug-interactions. The main drug interactions found in the drug-interaction analysis are shown in Table 5.\n\nSource: GOODMAN & GILMAN16, 2012; www.drugs.com21. AD, Alzheimer’s disease.\n\n\nDiscussion\n\nIn the present study, AD prevalence was higher in women [65.86% (n=27)]. This data corroborates Silva and collaborators (2012)17 results and may be justified by female longevity. Women tend to live longer than men, therefore, they spend more time of their lives with chronic diseases18.\n\nApproximately 80% of patients presented moderate and severe polypharmacy (Table 1). From 251 analyzed medications (corresponding 41 patients diagnosed with AD), the mean number of drugs taken was 7. Passareli and Filho (2007)19 showed a mean number of 6 drugs taken by AD patients, while other authors, such as Barbosa et al. (2008)20, demonstrated patients took approximately 8.6 medicaments concomitantly, indicating grave polypharmacy in this part of the population.\n\nDrug-interactions may occur for several reasons, such as pharmacokinetics, physiological antagonisms, additive effects, etc. The utilization of three drugs (donepezil, rivastigmine and memantine) to treat AD creates the false impression that controlling drug-interactions is simple. AD patients and caregivers are not aware of the interaction between drugs and enzymes. These enzymes may trigger inductive or inhibitory responses or serve as a substrate to other reactions.\n\nOnly pharmacokinetic interactions originated from AD drugs metabolism were utilized in the present study. A total of 30 possible drug-interactions between AChEI and other medications were identified. These interactions may be associated with increased risk and severity of ADRs, cumulative toxicity, medication errors, treatment adherence reduction, increase morbimortality and may also worsen patients’ cognitive functions22.\n\nRivastigmine is primarily metabolized through hydrolysis by esterase, but this drug does not appear to be a substrate for cytochrome P450 isozymes23,24,25. Therefore, drugs that modify the activities of isoenzymes do not alter kinetics characteristics of rivastigmine. When analyzing calcium channel antagonists, antidiabetics, non-steroidal anti-inflammatory drugs, antihistamines and anti-acids, no pharmacokinetic interaction with rivastigmine was found.\n\nHowever, the association of antihypertensive and beta-blockers with rivastigmine may contribute to additive effects that trigger bradycardia. Bradycardia might happen due to the block of beta-1-adrenergic receptors in the heart that, associated with acetylcholinesterase inhibition, cause an increase in acetylcholine levels, triggering a greater parasympathetic activity23,26,27.\n\nAssociation of antipsychotics and antidepressants with AChEI may inhibit the effects of AChEI, by the inhibition of cytochrome P450 2D6, which metabolizes AD medicaments. Due to this fact, the patient presents greater cognitive impairment23,28.\n\nDonepezil is also metabolized in the liver by cytochrome P450 isoenzymes 2D6 and 3A4. According to Pasquelati et al. (2015)28, an additive effect or a drug metabolism inhibition may occur when the cytochromes find specific substrates. These substrates may be, for example, antiarrhythmic (e.g., amiodarone) and antidepressant (e.g., Paroxetine, Perphenazine) drugs. In cases of such drug interactions, donepezil metabolism inhibition may potentiate the drug’s effects because donepezil’s active principles are, for a longer time, available in blood circulation.\n\nThe metabolism intensification and the decreased effects of donepezil may be observed with concomitant use of some antipsychotics (e.g., Quetiapine, Risperidone) or antidepressants (e.g. Sertraline), drugs that may be substrates to cytochrome P45029,30. Amiodarone, for example, may induce or retard AD drug metabolism, by being an antagonist and also a substrate of enzymes31.\n\nA common association of high-risk is the use of donepezil with no-steroidal anti-inflammatory agents, such as acetylsalicylic acid. The results of this interaction may be increased gastric acid secretion, and subsequently increased cholinergic activity, causing gastrointestinal hemorrhage32.\n\nMemantine is a weak base, excreted unchanged in urine; therefore, when analyzing its interaction with other drugs, urinary pH may be measured. Diuretics (e.g., Hydrochlorothiazide) increase body liquid elimination, which may cause a faster active principle clearance. When memantine and diuretics are taken together, blood concentrations of the diuretic may be reduced33.\n\nThe interaction of memantine with biguanide results in an activation of renal tubular excretion caused by metformin, increasing memantine clearance, which is similar to a diuretic effect34. However, no clinical study has been performed about this interaction.\n\nPrescriptions must be personalized according to the patients. In Brazil, if an AD patient uses SUS, they are influenced by the free of charge medications available in this system. Therefore, the list of medications dispensed by SUS should also be reevaluated and made adequate to elderly patients.\n\nAn ongoing evaluation of patients’ prescriptions is important since the majority of comorbidities begin between the 4th and 5th decade of life. At this age, comorbidities are treated with drugs appropriated to adults, but these medicaments are not changed when the individual reaches 60 years old9.\n\nIn summary, the present study verified drug-interactions that need particular attention, in order to improve the quality of life for the elderly population and decrease possible adverse effects. Some drug-interactions may begin after some years and are erroneously interpreted as a new disease, which complicates treatment and causes greater cognitive impairment in patients35.\n\nAChEI and NMDAR antagonist drugs interact with several drug classes (e.g., anxiolytics, antidepressants, antipsychotics, antihypertensive and antidiabetics), triggering polypharmacy. According to Hammes et al. (2008)36,37 polypharmacy is one of the leading causes of drug-interactions. Additionally, the authors reported that the risk of drug-interactions in patients who take eight or more medications increases by 100%.\n\nSeveral possible drug-interactions during AD treatment have been discussed by the present study. Consequently, a list of safe medications is indicated in Table 6 to treat AD patients with depression, anxiety, psychosis, hypertension, diabetes mellitus, cardiovascular diseases, inflammatory and fluid retention conditions, without interaction with AChEI and NMDAR antagonist drugs.\n\nSource: GOODMAN & GILMAN, 2012; www.drugs.com. AD, Alzheimer’s disease.\n\n\nConclusion\n\nThe present study showed the most frequent concomitant disease with AD were systemic arterial hypertension, depression, diabetes mellitus and hypercholesterolemia. To treat these concomitant diseases, patients took a mean of 7 medications daily, characterizing polypharmacy, which often triggers drug-interactions. The main pharmacological classes that result in drug-interaction were anxiolytics, antidepressants, antipsychotics, antihypertensives, and antidiabetics. Alternative treatments were proposed by the present study to replace PIM for elderly patients with AD.\n\nTo improve clinical safety, professionals must know the consequences of certain medications used in elderly patients, to identify these drugs and mainly, not to prescribe them. In Brazil, the implementation of a specific list in RENAME, including adequate drugs to elderly patients is necessary, as well as expanding the availability of these medications to elderly patients through the SUS.\n\nIn this study, polypharmacy was characterized in our patients. The mean number of drugs that they took was seven daily, used to treat concomitant diseases, which often trigger drug-interactions. We aimed to decrease these interactions and suggest drugs that no have interactions with concomitant disease.\n\n\nData availability\n\nRaw data for this article are available on OSF: http://doi.org/10.17605/OSF.IO/8UVR238.\n\nData are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThe Araucaria Foundation, CNPq, CAPES and AEPAPA.\n\n\nSupplementary material\n\nSupplementary File 1: Sociodemographic questionnaire.\n\nClick here to access the data.\n\nSupplementary File 2: Drug interactions. In this file are completely arranged, all interactions found of drugs ingested by patients. The analyzes were carried out through the literature GOODMAN AND GILMAN16 and also through the search in the database “drugs.com”, in which the drugs ingested were inserted individually and a report of the interactions was reported.\n\nClick here to access the data.\n\n\nReferences\n\nMirza Z, Beg MA: Possible Molecular Interactions of Bexarotene - A Retinoid Drug and Alzheimer’s Aβ Peptide: A Docking Study. Curr Alzheimer Res. 2017; 14(3): 327–334. PubMed Abstract \| Publisher Full Text\n\nZidan M, Arcoverde C, de Araújo NB, et al.: Alterações motoras e funcionais em diferentes estágios da doença de Alzheimer Motor and functional changes in different stages of Alzheimer’s disease. Rev Psiq Clín. 2012; 39(5): 3–7. Publisher Full Text\n\nSereniki A, Vital MABF: A doença de Alzheimer: aspectos fisiopatológicos e farmacológicos. Rev Psiquiatr. RS. 2008; 30(1 Supl). Publisher Full Text\n\nMazloomy MS, Soltani T, Morowatisharifabad MA, et al.: Activities of daily living and prevalence of chronic diseases among elderly people in Yazd. Toloo-e-Behdasht. 2014; 13(3(45)): 42–53. Reference Source\n\nSantana RS, Catanheide ID: Relação Nacional de Medicamentos: uma construção permanente. Cad Saúde Pública. 2015; 31(3): 647–650. Publisher Full Text\n\nPassarelli MCG: Medicamentos Inapropriados para Idosos: Um grave problema de Saúde Pública. Boletim informativo: Farmacovigilância. 2006. Reference Source\n\nCassoni TC, Corona LP, Romano-Lieber NS, et al.: [Use of potentially inappropriate medication by the elderly in São Paulo, Brazil: SABE Study]. Cad Saúde Pública. Rio de Janeiro. 2014; 30(8): 1708–1720. PubMed Abstract \| Publisher Full Text\n\nSecoli SR: [Polypharmacy: interaction and adverse reactions in the use of drugs by elderly people]. Universidade de São Paulo, São Paulo. Brasília, Rev Bras Enferm. 2010; 63(1): 136–40. PubMed Abstract \| Publisher Full Text\n\nRibeiro NP, Mascarenhas R, Mascarenhas MA, et al.: Polifarmácia utilizada por idosos residentes em instituições de longa permanência do município de Viamão/RS. Instituições de longa permanência do município de Viamão - Rio Grande do Sul. Revista Ciencia em Movimento. 2013. Publisher Full Text\n\nda Silva R, Schmidt OF, da Silva S: Polifarmácia em geriatria. Porto Alegre Revista da AMRIGS. 2012; 56(2): 164–174. Reference Source\n\nKusano LTE: Prevalência da polifarmácia em idosos com demência. 111 f. Dissertação (Mestrado em Ciências Médicas) - Universidade de Brasília, Brasília. 2009. Reference Source\n\nHolt S, Schmiedl S, Thürmann PA: Potentially inappropriate medications in the elderly: the PRISCUS List. Dtsch Arztebl Int. 2010; 107(31–32): 543–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBeers MH, Storrie M, Lee G: Potential adverse drug interactions in the emergency room. An issue in the quality of care. Ann Intern Med. 1990; 112(1): 61–4. PubMed Abstract \| Publisher Full Text\n\nMcKhann G, Drachman D, Folstein M, et al.: Clinical diagnosis of Alzheimer’s disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer’s Disease. Neurology. 1984; 34(7): 939–44. PubMed Abstract \| Publisher Full Text\n\nArevalo-Rodriguez I, Smailagic N, Roqué I Figuls M, et al.: Mini-Mental State Examination (MMSE) for the detection of Alzheimer’s disease and other dementias in people with mild cognitive impairment (MCI). Cochrane Database Syst Rev. 2015; (3): CD010783. PubMed Abstract \| Publisher Full Text\n\nGoodman & Gilman: As Bases Farmacológicas da Terapêutica. 12ª ed. Rio de Janeiro: McGraw-Hill, 2012.\n\nHebert LE, Scherr PA, McCann JJ, et al.: Is the risk of developing Alzheimer’s disease greater for women than for men? Am J Epidemiol. 2001; 153(2): 132–136. PubMed Abstract \| Publisher Full Text\n\nKuchemann BA: Envelhecimento populacional, cuidado e cidadania: velhos dilemas e novos desafios. Soc Estado. 2012; 27(1): 165–180. Publisher Full Text\n\nPassarelli MCG, Jacob-Filho W: Reações adversas a medicamentos em idosos: como prevê-las? Einstein. 2007; 5(3): 246–51. Reference Source\n\nBarbosa JAA, Belém LF, Sette IMF, et al.: Farmacoterapia adjuvante no tratamento da dor oncológica. Revista Brasileira em Promoção da Saúde. 2008; 21(2): 112–120. Publisher Full Text\n\nDrug Interactions Checker: Information from Drugs.com; c2000-10. 2017; Aceessd Jan 20, 2017. Reference Source\n\nRosa JS, Passos NM, Stefanon RM: Atenção Farmacêutica aos pacientes Hipertensos: Revisão de literatura. Escola superior de ciências da santa casa de misericórdia de Vitória - emescam, Vitória. 2016. Reference Source\n\nJann MW: Rivastigmine, a new-generation cholinesterase inhibitor for the treatment of Alzheimer’s disease. Pharmacotherapy. 2000; 20(1): 1–12. PubMed Abstract \| Publisher Full Text\n\nSpencer CM, Noble S: Rivastigmine. A review of its use in Alzheimer’s disease. Drugs Aging. 1998; 13: 391–411. PubMed Abstract\n\nJann MW, Shirley KL, Small GW: Clinical Pharmacokinetics and Pharmacodynamics of Cholinesterase Inhibitors. Clin Pharmacokinet. 2002; 41(10): 719–739. PubMed Abstract \| Publisher Full Text\n\nFerrari R, Cucchini F, Bolognesi R, et al.: How do calcium antagonists differ in clinical practice? Cardiovasc Drugs Ther. 1994; 8 Suppl 3: 565–575. PubMed Abstract \| Publisher Full Text\n\nPaulison B, Léos CL: Potential cardiotoxic reaction involving rivastigmine and beta-blockers: a case report and review of the literature. Cardiovasc Toxicol. 2010; 10(4): 306–310. PubMed Abstract \| Publisher Full Text\n\nPasqualetti G, Tognini S, Calsolaro V, et al.: Potential drug-drug interactions in Alzheimer patients with behavioral symptoms. Clin Interv Aging. 2015; 10: 1457–1466. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHiemke C, Pfuhlmann B: Interactions and monitoring of antipsychotic drugs. Handb Exp Pharmacol. 2012; (212): 241–265. PubMed Abstract \| Publisher Full Text\n\nBressler R: Grapefruit juice and drug interactions. Exploring mechanisms of this interaction and potential toxicity for certain drugs. Geriatrics. 2006; 61(11): 12–18. PubMed Abstract\n\nZhou SF, Xue CC, Yu XQ, et al.: Clinically important drug interactions potentially involving mechanism-based inhibition of cytochrome P450 3A4 and the role of therapeutic drug monitoring. Ther Drug Monit. 2007; 29(6): 687–710. PubMed Abstract \| Publisher Full Text\n\nThoonsen H, Richard E, Bentham P, et al.: Aspirin in Alzheimer’s disease: increased risk of intracerebral hemorrhage: cause for concern? Stroke. 2010; 41(11): 2690–2. PubMed Abstract \| Publisher Full Text\n\nWesemann W, Sonntag KH, Maj J: [Pharmacodynamics and pharmacokinetics of memantine]. Arzneimittelforschung. 1983; 33(8): 1122–1134. PubMed Abstract\n\nRao N, Chou T, Ventura D, et al.: Investigation of the pharmacokinetic and pharmacodynamic interactions between memantine and glyburide/metformin in healthy young subjects: a single-center, multiple-dose, open-label study. Clin Ther. 2005; 27(10): 1596–606. PubMed Abstract \| Publisher Full Text\n\nCahill JA: Responsibilities of physicians and pharmacists in preventing drug interactions. JAMA. 2002; 287(5): 586–587. PubMed Abstract\n\nHammes JÁ, Pfuetzenreiter F, Silveira Fd, et al.: Potential drug interactions prevalence in intensive care units. Rev Bras Ter Intensiva. 2008; 20(4): 349–354. PubMed Abstract \| Publisher Full Text\n\nVonbach P, Dubied A, Krähenbühl S, et al.: Prevalence of drug-drug interactions at hospital entry and during hospital stay of patients in internal medicine. Eur J Intern Med. 2008; 19(6): 413–20. PubMed Abstract \| Publisher Full Text\n\nBonini J: Raw data Polypharmacy in Alzheimer’s disease patients in Brazil: Guidance for pharmaceutical assistance. 2017. Data Source" }	[ { "id": "33054", "date": "30 Apr 2018", "name": "Fabio Monzani", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors presented a study aiming to evaluate the most frequent diseases concomitant with AD, analyze the interactions between medications and these diseases, and suggest a potentially safer alternative treatment for concomitant diseases. The topic is indeed very interesting, and of a crucial importance for the geriatric population. The work presented is potentially interesting, but unfortunately there are several major points to take into consideration.\nIn the definition of AD, I would include the tau deposition and the cortical atrophy as hallmark of the disease, together with the deposition of Amyloid β (Aβ). Moreover, I would mention about the amyloid deposition distribution pattern, which is more represented into the neocortex.\n\nThe use of the CDR scale should be better specified; if the score utilized to stratify the impairment is the “global” CDR or the sum of boxes and, should be referenced accordingly. (I assume it has been used the global, therefore I would suggest to reference Hughes et al. (1982)1.\n\nIn the session “data collection” the MMSE is explained as follows “The maximum score for MMSE is 30, and this indicates cognitive impairment”; considering that 30 in the MMSE is indicative of better performance, I would suggest to rephrase the sentence correctly.\n\n“Memantine must only be used during mild and moderate stages of AD” is not in agreement with the guidelines of using Memantine, which should be rather used in “moderate to severe dementia in Alzheimer’s Disease”\n\n“Therefore, knowing which medications interact with these drugs is fundamental to indicate the correct treatment for secondary diseases and, even, predict drug-interactions” I wouldn’t use the word “secondary” since the disease the authors are evaluating are concomitant and not “secondary” to AD.\n\nIn the discussion, when the authors write “AD patients and caregivers are not aware of the interaction between drugs and enzymes. These enzymes may trigger inductive or inhibitory responses or serve as a substrate to other reactions” it would be advisable to mention which enzymes/isoenzyme are they talking about (i.e Cytochrome P450)\n\nIn Table 5, I think it would be clearer if what the authors mean for “Drug opposite effect may worsen cognitive impairment, decreasing AChEI activity” will be more clearly specified.\n\nIn Table 6, some medications are suggested as “not interacting with Ache-I or NMDAR antagonists”. Considering that we are talking about a geriatric population, I don’t particularly agree with the definition of “safe”. For example, BDZ are considered safe for treating anxiety, according to the table. However, it is known that the CNS sensitivity to BDZs in increased, determining sedation at lower concentration. I would strongly recommend to carefully review this information, in light of the population taken into consideration in this paper, constituted not only by elderly, but demented.\n\nIs the work clearly and accurately presented and does it cite the current literature? Partly\n\nIs the study design appropriate and is the work technically sound? Partly\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-2068
https://f1000research.com/articles/6-2067/v1	29 Nov 17	{ "type": "Research Note", "title": "Prediction of influenza vaccine effectiveness for the influenza season 2017/18 in the US", "authors": [ "Slobodan Paessler", "Veljko Veljkovic", "Slobodan Paessler" ], "abstract": "Vaccination against seasonal influenza viruses is the most effective way to prevent infection. A key factor in the effectiveness of the seasonal influenza vaccine is its immunological compatibility with the circulating viruses during the season. The high evolutionary rate, antigenic shift and antigenic drift of influenza viruses, represents the main obstacle for correct prediction of the vaccine effectiveness for an upcoming flu season. Conventional structural and phylogenetic approaches for assessment of vaccine effectiveness have had a limited success in prediction of vaccine efficacy in the past. Recently, a novel bioinformatics approach for assessment of effectiveness of seasonal influenza vaccine was proposed. Here, this approach was used for prediction of the vaccine effectiveness for the influenza season 2017/18 in US.", "keywords": [ "H3N2", "influenza virus", "phylogenetic analysis", "seasonal influenza vaccine effectiveness" ], "content": "Introduction\n\nInfluenza vaccine effectiveness (VE) can vary from year to year, and among different age and risk groups. An important factor determining VE is the similarity or “match” between the influenza viruses used in the vaccine production and the viruses circulating in the community during the flu season. Evolution of influenza viruses between the time of vaccine selection and the beginning of the flu season (week 40 for the Northern Hemisphere) can seriously hamper VE. For this reason, in most years, the flu vaccine is 50% to 70% effective (http://www.cdc.gov/flu/professionals/vaccination/effectiveness-studies.htm; http://emergency.cdc.gov/HAN/han00374.asp), and in some years VE is <10%1.\n\nEach flu season researchers try to determine the VE adequately, but study results vary due to differences in study design, outcome(s) measured, population studied and the season in which the flu vaccine was studied. These differences make it difficult to compare the results of the several studies. However, in order to overcome these obstacles in determination of influenza VE, CDC established in 2004 the U.S. Flu Vaccine Effectiveness Network, which consists of five study sites across the United States. These study sites determine the VE by measuring the prevention of outpatient medical visits due to laboratory-confirmed influenza. The VE in the US for the period 2004–2015 determined by this Network is available.\n\nIn the 2017, flu season in Australia VE was only 10%2, which resulted in very high numbers of hospitalization and deaths among vaccinated patients. Most vulnerable was the elderly population of patients (>65 years). This low VE was ascribed to variability of the H3N2 influenza viruses that were dominant in Australia that season2.\n\nOur previous phylogenetic analysis of H3N2 influenza viruses, based on a novel bioinformatics algorithm, demonstrated how variability of this virus could seriously affect VE of seasonal influenza vaccines3. These results pointed out that close monitoring of evolution and spread of this virus is critical for prevention of a possible pandemic3.\n\nIn this study, we compared HA1 of H3N2 viruses isolated in US and Australia in the period July/September 2017. Presented results suggest that the flu season 2017/2018 in US will be not similar to that in Australia because the VE will be likely higher. Nevertheless, this situation could be changed if the minor group of viruses, which differ from the vaccine virus, would become dominant. For this reason, close monitoring of the evolution of H3N2 viruses in US during the flu season 2017/2018 is very important.\n\n\nMethods\n\nWe analyzed the hemagglutinin HA1 region of 251 human H3N2 viruses collected in Australia from July to September 2017 and 113 human H3N2 viruses collected in the US in the same period (non-redundant sequences are given in Supplementary File 1 and Supplementary File 2) that were stored in publicly open database GISAID (http://platform.gisaid.org). The vaccine virus A/Hong Kong/4801/2014 for 2017/2018 also is taken from this database.\n\nThe ISM is a virtual spectroscopy method for the study of the informational content of the protein primary structure. This method, which is described in detail elsewhere4, consists of two basic steps:\n\n(i) Representation of amino acids in the protein primary structure with their electron-ion interaction potential (EIIP)\n\n(ii) Conversion of the obtained numerical sequence into informational spectrum (IS) by Fourier transformation.\n\nFrequencies in IS represent information, which is encoded by the protein primary structure, and which determines its interaction with other proteins, DNA/RNA and small molecules.\n\nThe phylogenetic tree of the HA1 influenza proteins is generated with the ISM-based phylogenetic algorithm ISTREE. This algorithm was previously described in detail5 (for access to ISTRE we refer the reader to http://istree.bioprotection.org). Here, we used the ISM distance measure d defined on the specific frequency F = 0.299. This frequency was extracted by ISM as the common frequency component for HA1 of H3N2 influenza viruses3. The schematic presentations of calculated phylogenetic trees are given in Figure 1 and Figure 2. These trees in high resolution are given in Figure S1 and Figure S2.\n\nThis tree in high resolution is given in Figure S1.\n\nThis tree in high resolution is given in Figure S2.\n\n\nResults\n\nPreviously, it was shown that the IS frequency component F(0.299) is the informational hallmark of H3N2 viruses, which determines biological properties of HA1 of these influenza viruses3. We also showed that informational complementarity represented with F(0.299) between circulating influenza viruses and the vaccine virus correlate with VE of seasonal flu vaccine3.\n\nIn Figure 1 and Figure S1, the ISM-based phylogenetic tree is presented for HA1 from H3N2 viruses collected in Australia from July to September 2017 (Supplementary File 1). We constructed this tree using the amplitude on the frequency F(0.299) as a distance matrix of the HA1 sequences. In Figure 2 and Figure S2, we present the phylogenetic tree calculated for HA1 of 113 H3N2 viruses isolated in the US between July and September 2017.\n\nIn these phylogenetic trees, we included HA1 from the vaccine virus A/Hong Kong/4801/2014.\n\n\nDiscussion\n\nLow VE of influenza vaccine in Australia prompts the US and the European health authorities to prepare for the severe influenza season 2017/2018 with suboptimal VE on the Northern hemisphere2. On the other hand, there are no data or reported analysis of H3N2 viruses circulating in US in 2017 that would allow to predict the VE for the next flu season. Therefore, we performed the ISM-based phylogenetic analysis of H3N2 viruses representing precursors of seasonal flu viruses in the US. Result of this analysis served as the base for prediction of VE during the flu season 2017/2018 in the US.\n\nAs presented in Figure 1 and Figure 1S, all analyzes of Australian viruses generate two clusters and place the vaccine virus into the smaller group. This result suggests that vaccine would not be efficient against majority of Australian H3N2 viruses in the flu season 2017. This conclusion is in accord with reported low VE of influenza vaccine in Australia in 2017 flu season2.\n\nAs can be seen in Figure 2 and Figure 2S, the US H3N2 viruses also are grouped into two separate clusters, however, in contrast to Australian viruses, the vaccine virus A/Hong Kong/4801/2014 belongs to the largest cluster encompassing 71% of analyzed viruses. This result suggests that VE, at least in the beginning of this flu season will not be suboptimal in the US2. As for the prediction of VE in Australia, this could change during the flu season if some of the viruses from the small cluster would emerge. For this reason, continual monitoring of the evolution of H3N2 viruses is necessary.\n\nRecently, lack in effectiveness of influenza vaccine against H3N2 viruses has been attributed to mutation L194P in HA1, which is generated during the egg-based vaccine production process6. To test the effect of this mutation on the informational properties of HA1, we included this mutant of the vaccine virus in our analysis. As presented in Figure 2 and Figure 2S, this particular mutation is shifting the vaccine virus from the larger to the small cluster. This suggests that the vaccine carrying this mutation may protect only against smaller fraction of viruses during the flu season 2017/2018 and therefore result in low VE.\n\nIn summary, the presented results suggest that (i) the influenza vaccine will be effective against H3N2 viruses in the beginning of the flu season 2017/2018 in the US; (ii) it will be necessary to continue monitoring of evolution of H3N2 viruses during the flu season; and (iii) the vaccine with mutation L194P may have lower VE.\n\n\nData availability\n\nSequence data of the viruses were obtained from the GISAID EpiFlu™ Database. To access the database each individual user should complete the “Registration Form For Individual Users”. This form, together with detailed instructions, are available on the website. After submission of the Registration form, the user will receive a password. There are no any other restrictions for the access to GISAID. Conditions of access to, and use of, the GISAID EpiFlu™ Database and Data are defined by Terms of Use.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: The names of the human H3N2 influenza viruses collected in Australia from July to September 2017, taken from GISAID (http://platform.gisaid.org).\n\nClick here to access the data.\n\nSupplementary File 2: The names of the human H3N2 influenza viruses collected in US from July to September 2017, taken from GISAID (http://platform.gisaid.org).\n\nClick here to access the data.\n\nFigure S1: The ISM-based phylogenetic tree of HA1 from human H3N2 influenza viruses collected in Australia from July to September 2017.\n\nClick here to access the data.\n\nFigure S2: The ISM-based phylogenetic tree of the HA1 from human H3N2 influenza viruses collected in US from July to September 2017.\n\nClick here to access the data.\n\n\nReferences\n\nPebody RG, Warburton F, Ellis J, et al.: Low effectiveness of seasonal influenza vaccine in preventing laboratory-confirmed influenza in primary care in the United Kingdom: 2014/15 mid-season results. Euro Surveill. 2015; 20(5): 21025. PubMed Abstract\n\nSullivan SG, Chilver MB, Carville KS, et al.: Low interim influenza vaccine effectiveness, Australia, 1 May to 24 September 2017. Euro Surveill. 2017; 22(43): Pii=17-00707. PubMed Abstract\n\nVeljkovic V, Paessler S, Glisic S, et al.: Evolution of 2014/15 H3N2 Influenza Viruses Circulating in US: Consequences for Vaccine Effectiveness and Possible New Pandemic. Front Microbiol. 2015; 6: 1456. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nVeljkovic V, Paessler S: Possible repurposing of seasonal influenza vaccine for prevention of Zika virus infection [version 2; referees: 2 approved]. F1000Res. 2016; 5: 190. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPerovic V: Novel algorithm for phylogenetic analysis of proteins: application to analysis of the evolution of H5N1 influenza viruses. J Mathem Chem. 2013; 51: 2238–2255. Publisher Full Text\n\nWu NC, Zost SJ, Thompson AJ, et al.: A structural explanation for the low effectiveness of the seasonal influenza H3N2 vaccine. PLoS Pathog. 2017; 13(10): e1006682. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "28515", "date": "18 Dec 2017", "name": "Richard A. Bowen", "expertise": [ "Reviewer Expertise Virology (including influenza) and animal models" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very interesting report describing a technique for computational assessment of potential vaccine efficacy of H3N2 influenza viruses. It is clear that improved techniques for predicting seasonal influenza virus vaccine efficacy are needed and this could become one of a group of evaluations that yield more precise predictions.\nI am not competent to evaluate the mathematical underpinnings of these analyses, but it would seem like some statistical analyses should be invoked; I leave it to the other reviewer(s) and editors to indicate if that is the case. The other comment I would make is that it seems as if a more historical set of evaluations would be valuable - the 2017 season is not yet over and, as the authors repeatedly indicate, things make change in terms of the viruses that come to predominate, especially in the U.S.\nOverall, I think this is a valuable contribution to forecasting for influenza virus efficacy and encourage the authors to use additional historical datasets for further evaluate the utility of the technique.\n\nMinor comments:\nIn the 2017, flu season in Australia VE was only 10% -> In the 2017 flu season in Australia, VE was ..\nIn this study, we compared HA1 of H3N2: you haven't defined HA1 yet - might say the sequence of hemagglutinin HA1\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Partly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "30129", "date": "01 Feb 2018", "name": "Igor S. Lukashevich", "expertise": [ "Reviewer Expertise Medical virology", "vaccine R&D" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is very timing report addressing the most important public health issue, prediction of influenza vaccine efficacy. While we certainly are better prepared to flu epidemics in 2018 than in 1918, low efficiency of vaccination in recent flu season in Australia and currently ongoing flu in the US with increasing numbers of severe cases among children and elderly patients raised reasonable questions regarding prediction of correct vaccine formulations. Influenza viruses are moving targets and we have to do better job to predict and manufacture effective vaccines in timing manner. The authors applied innovative Informative Spectrum Method and analysed an extensive collection of influenza isolates from Australia and the US during July – September 2017. Based on this analysis they have predicted sub optional vaccine effectiveness for ongoing flu season in the US. The manuscript is well-written, results are solid and supported by well-developed bioinformatics tools. Discussion is appropriate. The manuscript is recommended for accelerated publication.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Yes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2067
https://f1000research.com/articles/6-2066/v1	29 Nov 17	{ "type": "Research Article", "title": "Out-of-pocket health expenditure and fairness in utilization of health care facilities in Cambodia in 2005 and 2010", "authors": [ "Koustuv Dalal", "Olatunde Aremu", "Gainel Ussatayeva", "Animesh Biswas", "Koustuv Dalal", "Olatunde Aremu", "Gainel Ussatayeva" ], "abstract": "Background: Out-of-pocket (OOP) payments for health care are highly pervasive in several low-and-middle income countries. The Cambodian health system has envisaged massive repositioning of various health care financing to ensure equitable access to health care. This analysis examines catastrophic, economic, as well as fairness, impacts of OOP health care payments on households in Cambodia over time. Methods: Data from two waves of a nationally representative household survey conducted in Cambodia (CDHS Surveys 2005 and 2010) were utilized. Healthcare utilizations based on economic status were compared during 2005 and 2010. Variables of interests were i) where care was sought and the instances of treatments, i.e. was treatment sought the first, second or third time; (ii) the mode of payment for treatment of the respondent or for any household member due to sickness or injury in the last 30 days prior to the survey period. Lorenz curves were applied to assess the degree of distribution of inequality in OOP expenditures between different income brackets. Results: The findings revealed that there was inequality and unfairness in health care payments, and catastrophic spending is more common among the poor in Cambodia. The majority of people from poorer households experienced economic hardship and have taken to catastrophic health care spending through sales of personal possessions. Conclusion: Based on the findings from this analysis, more attention is needed on effective financial protection for Cambodians to promote fairness. The government should increase spending on services being provided at public health care facilities to reduce ever increasing reliance on private sector providers. These approaches would go a long way to reduce the economic burden of care utilization among the poorest.", "keywords": [ "Equity", "Fairness", "Out-of-pocket payment", "Health Expenditure", "Low-income countries", "Cambodia" ], "content": "Introduction\n\nGlobally, the main goal of a health care system is to have appropriate funding mechanisms for individuals to acquire care for preventive and curative health needs without deepening into poverty. There is a general consensus that ease of access to health care facilities has the potential to improve the health and wellbeing of individuals and increase the life expectancy of the entire population1,2. However, the pluralistic nature and differences in financing mechanisms of most health systems around the world means varied methods will be adopted by policy makers for health care provision. Revenue collection through general taxation and compulsory social health insurance, as well as individual private health insurance schemes, are among the most commonly used methods for funding health care provisions in high income nations3. Sadly, in spite of the successes of these funding strategies in high income nations, inadequate planning and lack of political will has given rise to non-existence of universal financing mechanisms in low-and-middle-income countries (LMICs). In these countries, many individuals continue to rely on out-of-pocket (OOP) payments each time they access care, and as a result, are not shielded from economic hardship due to huge health care expenditures. Catastrophic health expenditure through OOP payments is a global phenomenon and can exist regardless of health care funding mechanisms. However, OOP payments constitute a significant component of health spending in most LMICs. According to a study investigating global estimates of catastrophic spending and OOP payments4, approximately 150 million people suffer financial burden each year due to health care payments, and about 100 million are pushed into poverty because of OOP payments.\n\nPaying for health care through OOP may prevent patients from seeking medical care when needed due to economic constraints. This is particularly common for people with lower incomes5. Low income groups sometimes risk debt due to paying for health care6. In Cambodia, as is the case with most countries in South East Asia6–9, excessive reliance on OOP payment for care is pervasive due to lack of universal health coverage10. Cambodia is a country with a turbulent history, enduring genocide and societal collapse, but is now rapidly evolving and has managed to decrease poverty significantly11. The repositioning of the Cambodian health system is aimed at preventing and controlling communicable and non-communicable diseases12,13. Although the health care system has also experienced progress, it still is in need of further development, as the majority of health expenditure comes from OOP payments and this usually benefits unregulated private health care14. There was a health care system reform in 1996, and the National Charter on Health Financing was introduced allowing user fees in health care facilities15. In conjunction with this charter on health financing, a health equity fund was introduced in 2009 to aid access to health care for the poor. However, the health equity fund has not been implemented on a national scale, but rather has been used in different ways in different districts16,17. Examining the impact of catastrophic health care spending on household impoverishment during the introduction of a new funding mechanism, such as health equity fund, over a period of time is of policy relevance. While several studies have been conducted to assess the impact of catastrophic expenditure on household impoverishment over a period of time in other countries in South East Asia6–10, no such study have been done in Cambodia.\n\nThe present study was conducted to bridge this research gap and examine, for the first time, trends in catastrophic OOP payment burden and equity in use of health care facilities among a nationally representative sample of Cambodian adults aged 15–49 across 611 communities. Specifically, this study aimed to: first, examine fairness in the distribution of cost burden among wealth strata and compare trends between 2005 and 2010; and second, characterise sources of OOP payment for treatment and compare the trends between 2005 and 2010. Understanding such comparisons would aid in further planning and future implementation of various health benefit packages aimed at alleviating high OOP payments for healthcare use in Cambodia.\n\n\nMethods\n\nSecondary data analysis was conducted using publicly available individual and community level data from the Cambodian Demographic and Health Surveys (CDHS) of 2005 and 201018,19. These survey data were collected from nationally representative samples of households. Approval to use the data was granted by The DHS Program, Rockville, USA. DHS are a series of population-based surveys commonly conducted in most LMICs by in-country national agencies under the technical assistance of ICF Macro with financial support from USAID.\n\nBriefly, the CHDS employs a two-stage sampling procedure, with the first stage involving the selection of primary sampling units (PSUs); these are probability proportional to size and represent the number of households within the PSU. The second stage uses a systematic technique to sample households from each of the selected PSUs units. The full details of the methods and procedure used in data collection in CDHS surveys is provided elsewhere19. A total of 8578 weighted sample of adults from two surveys was distributed as follows, which were used for analysis: 2589 in 2005 survey; 5989 in 2010 survey\n\nThe outcome variables for this analysis were: (i) where care was sought and the instances of treatments, i.e. was treatment sought the first, second or third time; (ii) the mode of payment for treatment of the respondent or for any household member due to sickness or injury in the last 30 days prior to the survey period. First, to estimate the distribution of economic hardship among wealth groups, principal component analysis (PCA) was used20. PCA was used to calculate an asset index for each household using the respondent response about possession of a set of household assets. Second, the resulting index based on economic hardship was then used to rank households into quintiles as poorest, poorer, middle, richer and richest18–20. Individuals who live below the poverty line are determined from their households in the poorest quintile18–20. The derived wealth quintiles was used as the only dependent variable in this analysis.\n\nDescriptive statistics was adapted to characterise healthcare utilization by the respondents. Lorenz curves were applied to assess the degree of distribution of inequality in OOP expenditures. The Lorenz curve is a measure of the distribution of wealth within a population21–23. Briefly, the X axis on the curve denotes the percentile of the population distributed according to the characteristic under observation. The observed characteristic in this analysis is the economic status, as a proxy of income19,20. The (y) value of the curve represents the exact proportion of the overall value of costs accrued to people that are no wealthier than a specified estimated percentile of the population. For better understanding, we have used three different costs. These are cost of the treatment, transport cost and total costs,\n\nThe Lorenz curve can be expressed mathematically as shown belows\n\n\n\nWhere F (y) represents the cumulative distribution functions of ordered individuals and μ represents the average size. The Lorenz curve, in the simplest form, is usually interpreted as follows: if there is no difference among all individuals under observation, the Lorenz curve would be a straight diagonal line, called the line of equality. However, if there is an existence of inequality among the population, then the Lorenz curve would show a fall below the line of equality23. All the analyses were conducted using STATA 13 software package.\n\n\nResults\n\nA total of 5989 participants participated in the 2010 study. Overall, 92% sought health services for a first-time treatment in 2010 compared to 90% in 2005. For services sought for second treatments, there was a decrease from 29% in 2005 to 24% in 2010. Similar results were shown for third treatments; 40% in 2005 to 33% in 2010.\n\nTable 1 depicts the information of service utilization for the 1st, 2nd and 3rd instances of treatments for illness or injury. In 2005, almost 83% of people belonging to the household 20% below the poverty line (i.e. poorest quintile) sought treatment at the 1st instance compared with 95% of those from rich household. There is an increase in the proportion of people from the poorest strata seeking treatment at the 1st instance in 2010 (i.e. from 83% to 90%), but the number of the richest seeking treatment at the 1st instance between 2005 and 2010 remains the same. There is, however, a significant reduction in the percentage of both the poorest and the richest that sought treatment in the 2nd and 3rd instances between 2005 and 2010; for example, 28% of both the poorest and richest sought treatment at the 2nd instance in 2005, by 2010, the percentage of poorest people seeking treatment for the 2nd instance fell to 23%. The analysis also revealed a decline to 20% in 2010 for rich people seeking care. For the 3rd instance and in 2005, more people, 45% from the poorest households used heath care more than their rich counterparts (35%). In 2010, whilst the percentage of rich people seeking care for the 3rd instance remained unchanged from that of 2005 and much lowered compared to those from poorest households, there is a decline in the percentage of poorest people (34%) seeking care in 2010 compared with 2005. Almost 90% and 70% of people in both the 20% below (poorest quintile) and 20% above (poorer quintile) the poverty line have in 2005 and 2010, respectively, have used more health care at the 3rd instance than their rich counterparts (33%) for both years.\n\nThere was an increase in services sought for 1st treatment among all groups, except for the richest group, who remained the same, between 2005 and 2010. For services sought for 2nd treatment, there was a decrease in all groups. Regarding health care utilization for 3rd treatment, a similar decrease was observed; however this was not significant in 2005 or 2010.\n\nOverall, there was an increase in utilization of both public and private health care facilities between 2005 and 2010, as can be seen in Figure 1. There was an increase in usage of private facilities as the preferred place for service utilization at 1st instance i.e. from 38.6% in 2005 to 61% in 2010. There was also an increase, albeit a small one, in use of public facilities, 26.5% in 2005 to 32.6% in 2010. For utilization of health care at the 2nd instance, similar results can be found; increase in usage of private health care from 37.7% in 2005 to 63.7% in 2010 and increase in usage of public health care from 26.7% in 2005 to 29.4 in 2010. This is also the same for the 3rd instance; an increase in utilization of private health care facilities from 37% in 2005 to 64.8% in 2010. There was been a decrease in usage of informal health care facilities (such as shop/market, magician, religious leader, all characterised as others), for all three different instances from 2005 to 2010. These results who that, generally, a higher percentage of Cambodians have a strong preference for private health care facilities.\n\nMode of out-of-pocket payment. Figure 2 displays a breakdown of percentages of different modes of OOP. Briefly, the mode of OOP was divided into the following categories: wages/pocket money, savings, sale of assets, interest loan, non-interest loan, and other sources, e.g. such as help from an NGO. In 2005, 44% of all OOP payments came from wages/pocket money, followed by 33% from savings. In 2010, 31% of OOP payments were from wages/out of pocket money and 41% from savings. Payments from loans generally remained the same between 2005 and 2010, around 5-7%. Sale of assets as means to pay for OOP payments for health care increased from 3% in 2005 to 6% in 2010. Gifts from relatives and friends (5% in 2005 and 6% in 2010) were also OOP sources. The health equity fund introduced in 2009 seems to have effect on payments and accounted for 2% of the OOP payments in 2010.\n\nTransport cost, treatment cost and total cost of treatment. Degree of fairness in terms of spending on transport, treatment and total cost of treatment was examined using Lorenz curves (Figures 3a–c). As depicted in Figure 3a, for all categories of treatment instances, the transportation cost is the most equal across nearly all the economic strata, with individuals in the poorest quintiles of the economic strata being more able to cope with spending. Compared to 2005, in 2010 individuals in the 3rd, 4th and 5th strata are in a relatively good condition to bear the cost of transportation. Regarding the treatment cost, Figure 3b indicates that inequality was more pronounced in 2010 compared to 2005. For instance, people at the 4th & 5th quintile of the economic strata are in relatively good position to afford the cost of treatment than in 2005 for all the instances of treatment. Figure 3c shows that for all instances of treatments, the total cost is most unequal in 2005 compared to 2010. For instance, those at the 4th & 5th quintiles of the economic strata are more able to bear total treatment costs relative to those at the lower strata.\n\nLorenz curves for Transport cost (3a), Treatment cost (3b) and Total cost of treatment (3c): comparison between 2005 and 2010.\n\n\nDiscussion\n\nThis study provides the first detailed trend analysis of catastrophic OOP health expenditure and fairness of healthcare facilities utilization in Cambodia in 2005 and 2010. The findings show that the trend of catastrophic spending and fairness in utilization of health care facilities is not improving. The finding that more people from the poorer households are seeking treatment in all three instances compared to richer households over the two periods (2005 and 2010) is not new. The reason for this may be because individuals from lower strata of the economic index tend to patronize hawkers (here, a kind of quack without any medical degree selling medicine), as these providers are known to charge nominal fees24,25. Care seeking from hawkers and unqualified health care professionals has been shown to be a precursor for treatment failures and switching of care providers at private and public health facilities24,26,27. The present finding supports a report from elsewhere in South East Asia, i.e. that poorer individuals have large healthcare burdens28.\n\nThe present results displayed a huge increase in utilization of private facilities compared to public facilities from 2005 to 2010. Hence, one may argue that costs are an important determinant of choice of place of treatments. People would use more public facilities to avoid healthcare burden at private facilities. In this study, however, the trend in use of health care facilities has been shown to tilt consistently towards the use of private facilities for all instances of service use for the two time periods. This finding is not new, but is consistent with what has been reported before in other LMICs, and South East Asia in particular4,28. Countries in South East Asia have witnessed an increase in the proliferation of private health care facilities in the last decade. One probable reason for the present observed finding may be one of the unique features of poor health systems, where private and public care facilities always substitute each other, mostly in LMICs29. In addition to this, inefficiency in service delivery in public health systems has been shown to be another contributory factor. For example, long waiting times, nonchalant attitudes of health care workers and perennial lack of essential medicines, coupled with informal fees charged by medical personnel to survive poor wages30–32.\n\nFinancing health care through formal and informal OOP payments have proven to affect people’s behavior in seeking care, especially those from poor households27,33. In this analysis, the pattern of OOP has been shown to be consistent and comprised of loans both with and without interests, wages/pocket money and savings in both 2005 and 2010. This finding supports what has been reported in several studies conducted across different countries34–36. The present analysis documents a wide variation in the mode of payment for health care between the two periods in relation to use of personal savings and wages/pocket money. Evidence has consistently shown that an individual from a poorer household faces a huge economic burden from health care costs than their richer counterpart36. People saved more in 2010 and relied less on their wages and pocket money to offset payments for their treatment compared to 2005. Another explanation for this observation could be related to people’s awareness of the importance of setting aside a smaller portion of their income for health care emergencies to avoid selling and borrowing.\n\nHealth equity funds, exemption schemes and other subsidizations have been introduced to address inequity of health care utilization, which target the poor, in several LMICs. It can be difficult to identify and target the intended people for these exemption schemes and other subsidizations; however, studies in Cambodia found that the health equity fund to be rather successful in this country17. In contrast, in the present study, the health equity fund only accounted for 2% of the OOP payments being made. Possession of health insurance has been shown to hold promise for financing individual health care costs in LMICs37. The use of health insurance as a mode of payment for health service utilization was reported in 2010, but not in 2005. This finding is in line with the results from other studies38,39.\n\nEconomic hardship experienced by people from poorer households when accessing health care is immeasurable. As in other studies, the present analysis further documents the existence of inequity and unfairness in cost burden between the poor and the rich in Cambodia when accessing care. In summary, the distribution of cost burden and catastrophic spending among Cambodian adults was more inequitable over the two time periods. However, there is a growing understanding of the need to bridge poor-rich inequalities in access to health care through adoption of several coping mechanisms among the entire populace.\n\nA health system should, according to the WHO, improve the health of the population they serve, respond to people’s expectations and provide financial protection against the costs of ill-health2. With the present reported result, Cambodian health systems, which heavily rely on OOP payments, do not meet the last objective. Furthermore, according to the Strategic Plan 2008–2015 from the Ministry of Health, one of five working principles are “social health protection, especially for the poor and vulnerable groups”15. The findings from this analysis echoes the need to ensure that those from poorer households are protected against hardship spending when seeking care. As such, more work is needed to guarantee access to sufficient health care for the poor and vulnerable in Cambodia. One of the ways to mitigate financial hardships constantly being faced by those from poorer households and reduce poverty drift through catastrophic health care spending is to adopt point-of-care health financing mechanisms targeted at those in need40.\n\nThis study had several important limitations that warrant mentioning. First, this analysis is based on existing survey data; hence it is difficult to ascertain the long-term effect of borrowing on individuals that may lead to economic hardship. Second, it is difficult to account for multiple modes of payments for health care use at individual levels. Third, this analysis used an asset-based index as a proxy measure of economic ability at household level and may be subject to criticism. However, an asset–based index as a surrogate measure for household wealth has been shown to be the most appropriate in LMICs20.\n\nFindings from this analysis corresponds with the results of other studies and asserts the need for governments in LMICs to adopt a pro-poor funding mechanism. Although the introduction of an health equity fund is a welcome idea, Cambodian policy makers should take a cue from other LMICs and adopt multiple means tested health care financing option, such as community based health insurance and social insurance, to help mitigate financial hardship due to OOP payments41–43. In addition, the government should increase spending on services being provided at public health care facilities to reduce ever increasing reliance on private sector providers. These approaches would go a long way to reduce the economic burden of care utilization among the poorest.\n\n\nData availability\n\nThe DHS Program owns data used in this study. The DHS for Cambodia 2005 and 2010 are available for researchers interested in further analyses. Researchers should contact the DHS Program and to get permission to use the required data (https://dhsprogram.com/data/Access-Instructions.cfm).", "appendix": "Competing interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nBunker JP, Frazier HS, Mosteller F: Improving health: measuring effects of medical care. Milbank Q. 1994; 72(2): 225–258. PubMed Abstract \| Publisher Full Text\n\nWHO: The world health report 2000 - Health systems: improving performance. Geneva: World Health Organization; 2000. Reference Source\n\nSaksena P, Xu K, Evans DB: Impact of out-of-pocket payments for treatment of non-communicable diseases in developing countries: A literature review. Geneva: World Health Organization. 2011. Reference Source\n\nXu K, Evans DB, Carrin G, et al.: Protecting households from catastrophic health spending. Health Aff (Millwood). 2007; 26(4): 972–983. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAremu O, Lawoko S, Dalal K: Neighborhood socioeconomic disadvantage, individual wealth status and patterns of delivery care utilization in Nigeria: a multilevel discrete choice analysis. Int J Womens Health. 2011; 3: 167–174. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGarcia-Diaz R, Sosa-Rubi SG: Analysis of the distributional impact of out-of-pocket health payments: evidence from a public health insurance program for the poor in Mexico. J Health Econ. 2011; 30(4): 707–718. PubMed Abstract \| Publisher Full Text\n\nNguyen CV: The impact of voluntary health insurance on health care utilization and out-of-pocket payments: new evidence for Vietnam. Health Econ. 2012; 21(8): 946–966. PubMed Abstract \| Publisher Full Text\n\nLee WY, Shaw I: The impact of out-of-pocket payments on health care inequity: the case of national health insurance in South Korea. Int J Environ Res Public Health. 2014; 11(7): 7304–7318. 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PubMed Abstract \| Publisher Full Text" }	[ { "id": "28958", "date": "12 Dec 2017", "name": "Kazi Selim Anwar", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFindings of this important study highlighted that the Combodian Govt. needs effective financial protection plan to promote spending on health-care-utilization. It would reduce economic burden of health-care utilization among the poorest communities. This research has high potential to allow a platform of replicating this study by others in LMICs.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "28781", "date": "04 Jan 2018", "name": "Aziz Rahman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nFindings of this study add further evidence regarding the burden of healthcare expenditure amongst the poor people in a developing country like Cambodia.\nThe authors have explained the rationale for conducting this study well and utilized secondary database to answer the research question. Data comparison between 2010 and 2005 in the Figure and Table were informative. It was good to observe the changes regarding use of other facilities in the third instance in 2010 compared to the data from 2005. However, use of private facilities in the second instance did not change over years, which was not an unexpected scenario in developing countries although not acceptable. Hence policy makers should address the factors to increase utilization of public healthcare facilities. The major portion of the out of pocket expenditure was from either savings or wages/pocket money, which did not change over years as well.\nThe authors have nicely indicated policy implications of the evidence generated from this study, which should be communicated to the policy makers in Cambodia to expect changes in future.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nI cannot comment. A qualified statistician is required.\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2066
https://f1000research.com/articles/6-1151/v1	20 Jul 17	{ "type": "Review", "title": "A multi-disciplinary perspective on emergent and future innovations in peer review", "authors": [ "Jonathan P. Tennant", "Jonathan M. Dugan", "Daniel Graziotin", "Damien C. Jacques", "François Waldner", "Daniel Mietchen", "Yehia Elkhatib", "Lauren B. Collister", "Christina K. Pikas", "Tom Crick", "Paola Masuzzo", "Anthony Caravaggi", "Devin R. Berg", "Kyle E. Niemeyer", "Tony Ross-Hellauer", "Sara Mannheimer", "Lillian Rigling", "Daniel S. Katz", "Bastian Greshake Tzovaras", "Josmel Pacheco-Mendoza", "Nazeefa Fatima", "Marta Poblet", "Marios Isaakidis", "Dasapta Erwin Irawan", "Sébastien Renaut", "Christopher R. Madan", "Lisa Matthias", "Jesper Nørgaard Kjær", "Daniel Paul O'Donnell", "Cameron Neylon", "Sarah Kearns", "Manojkumar Selvaraju", "Julien Colomb", "Jonathan M. Dugan", "Daniel Graziotin", "Damien C. Jacques", "François Waldner", "Daniel Mietchen", "Yehia Elkhatib", "Lauren B. Collister", "Christina K. Pikas", "Tom Crick", "Paola Masuzzo", "Anthony Caravaggi", "Devin R. Berg", "Kyle E. Niemeyer", "Tony Ross-Hellauer", "Sara Mannheimer", "Lillian Rigling", "Daniel S. Katz", "Bastian Greshake Tzovaras", "Josmel Pacheco-Mendoza", "Nazeefa Fatima", "Marta Poblet", "Marios Isaakidis", "Dasapta Erwin Irawan", "Sébastien Renaut", "Christopher R. Madan", "Lisa Matthias", "Jesper Nørgaard Kjær", "Daniel Paul O'Donnell", "Cameron Neylon", "Sarah Kearns", "Manojkumar Selvaraju", "Julien Colomb" ], "abstract": "Peer review of research articles is a core part of our scholarly communication system. In spite of its importance, the status and purpose of peer review is often contested. What is its role in our modern digital research and communications infrastructure? Does it perform to the high standards with which it is generally regarded? Studies of peer review have shown that it is prone to bias and abuse in numerous dimensions, frequently unreliable, and can fail to detect even fraudulent research. With the advent of Web technologies, we are now witnessing a phase of innovation and experimentation in our approaches to peer review. These developments prompted us to examine emerging models of peer review from a range of disciplines and venues, and to ask how they might address some of the issues with our current systems of peer review. We examine the functionality of a range of social Web platforms, and compare these with the traits underlying a viable peer review system: quality control, quantified performance metrics as engagement incentives, and certification and reputation. Ideally, any new systems will demonstrate that they out-perform current models while avoiding as many of the biases of existing systems as possible. We conclude that there is considerable scope for new peer review initiatives to be developed, each with their own potential issues and advantages. We also propose a novel hybrid platform model that, at least partially, resolves many of the technical and social issues associated with peer review, and can potentially disrupt the entire scholarly communication system. Success for any such development relies on reaching a critical threshold of research community engagement with both the process and the platform, and therefore cannot be achieved without a significant change of incentives in research environments.", "keywords": [ "Open Peer Review", "Social Media", "Web 2.0", "Open Science", "Scholarly Publishing", "Incentives", "Quality Control" ], "content": "1 Introduction\n\nPeer review is the process in which experts are invited to assess the quality, novelty, validity, and potential impact of research by others, typically while it is in the form of a manuscript for an article, conference, or book (Spier, 2002). For the purposes of this article, we are exclusively addressing peer review in the context of manuscripts for research articles, unless specifically indicated; different forms of peer review are used in other contexts such as hiring, promotion, tenure, or awarding research grants (see, e.g., Fitzpatrick, 2011b, p. 16). Peer review comes in various flavors that result from different approaches to the relative timing of the review (with respect to article drafting, submission, or publication) and the transparency of the process (what is known to whom about submissions, authors, reviewers and reviews) (Ross-Hellauer, 2017). The criteria used for evaluation, including methodological soundness or expected impact are also important variables to consider. In spite of the diversity of the process, it is generally perceived as the gold standard that defines scholarly publishing by researchers and the wider public alike, and often deemed the primary determinant of scientific, theoretical, and empirical validity (Kronick, 1990). Consequently, peer review is a vital component at the core of research communication processes, with repercussions for the very structure of academia, which largely operates through a peer reviewed publication-based reward and incentive system (Moore et al., 2017). However, peer review is applied inconsistently both in theory and practice (Pontille & Torny, 2015), and generally lacks any form of transparency or formal standardization. As such, it remains difficult to know what we actually mean when we identify something as a “peer reviewed publication.”\n\nTraditionally, the function of peer review has been as a vetting procedure or gatekeeper to assist the distribution of limited resources—for instance, space in peer reviewed print publication venues, research time at specialized research facilities, or competitive research funds. Nowadays, it is also used to assess whether and how a given piece of research fits into the overall body of existing scholarly knowledge, and which journal it is suitable for and should appear in. This has consequences for whether the body of published research produced by an individual merits consideration for a more advanced position within academic or industrial research. With the advent of the Internet, the physical constraints on distribution are no longer present, and, at least in theory, we are now able to disseminate research content rapidly and at relatively negligible cost (Moore et al., 2017). This has led to the increasing popularity of digital-only publication venues that vet submissions based on the soundness of the research (e.g., PLOS, PeerJ). Such a flexibility in the filter function of peer review reduces, but does not eliminate, the role of peer review as a selective gatekeeper. Due to such innovations, ongoing discussions about peer review are intimately linked with contemporaneous developments in Open Access (OA) publishing and to broader changes in open research (Tennant et al., 2016).\n\nThe goal of this article is to investigate the historical evolution in the theory and application of peer review in a socio-technological context. We use this as the basis to consider how specific traits of consumer social Web platforms can be combined to create an optimized hybrid peer review model that is more efficient, democratic, and accountable than the traditional process.\n\nAny discussion on innovations in peer review must take into account its historical context. By understanding the history of scholarly publishing and the interwoven evolution of peer review, we recognize that neither are static entities, but in fact covary with each other, and therefore should be treated as such. By learning from historical experiences, we can also become more aware of how to shape future directions of peer review evolution and gain insight to what the process should look like in an optimal world. The actual term “peer review” only appears in the scientific press in the 1960s. Even in the 1970s, it was associated with grant review and not with evaluation and selection for publishing (Baldwin, 2017a). However, the history of evaluation and selection processes for publication clearly predates the 1970s.\n\n1.1.1 The early history of peer review. The origins of scholarly peer review of research articles are commonly associated with the formation of national academies in 17th-century Europe, although some have found foreshadowing of the practice (Al-Rahawi, c900; Spier, 2002). We call this period the primordial time of peer review (Figure 1). Biagioli (2002) described in detail the gradual differentiation of peer review from book censorship, and the role that state licensing and censorship systems played in 16th-century Europe; a period when monographs were the primary mode of communication. Several years after the Royal Society of London (1660) was established, it created its own in-house journal, Philosophical Transactions; around the same time, Denis de Sallo published the first issue of Journal des Sçavans. Both of these journals were first published in 1665. In London, Henry Oldenburg was appointed Secretary to the Royal Society and became the founding editor of Philosophical Transactions. Here, he took on the role of gathering, reporting, critiquing, and editing the work of others, as well as initiating the process of peer review as it is now commonly performed (Manten, 1980; Oldenburg, 1665). Due to this origin, peer review emerged as part of the social practices of gentlemanly learned societies. These social practices also included organizing meetings and arranging the publications of society members, while being responsible for editorial curation, financial protection, and the assignment of individual prestige (Moxham & Fyfe, 2016). The development of these prototypical scientific journals gradually replaced the exchange of experimental reports and findings through correspondence, formalizing a process that had been essentially personal and informal until then. “Peer review”, during this time, was more of a civil, collegial discussion in the form of letters between authors and the publication editors (Baldwin, 2017b). Social pressures of generating new audiences for research, as well as new technological developments such as the steam-powered press, were also crucial. The purpose of developing peer reviewed journals became part of a process to deliver research to both generalist and specialist audiences, and improve the status of societies and fulfil their scholarly missions (Shuttleworth & Charnley, 2016).\n\nThe interactive data visualization is available at https://dgraziotin.shinyapps.io/peerreviewtimeline, and the source code and data are available at https://doi.org/10.6084/m9.figshare.5117260 (Graziotin, 2017).\n\nFrom these early developments, the process of independent review of scientific reports by acknowledged experts gradually emerged. However, the review process was more similar to non-scholarly publishing, as the editors were the only ones to appraise manuscripts before printing (Burnham, 1990). As early as 1731, the Royal Society of Edinburgh adopted a formal peer review process in which materials submitted for publication in Medical Essays and Observations were vetted and evaluated by additional knowledgeable members (Kronick, 1990; Spier, 2002). In 1752, the United Kingdom’s Royal Society created a “Committee on Papers” to review and select texts for publication in Philosophical Transactions (Fitzpatrick, 2011b, Chapter One). The primary purpose of this process was to select information for publication to account for the limited distribution capacity, and remained the authoritative purpose of peer review for more than two centuries.\n\n1.1.2 Adaptation through commercialisation. Through time, the diversity, quantity, and specialization of the material presented to journal editors increased. This made it necessary to seek assistance outside the immediate group of knowledgeable reviewers from the journals’ sponsoring societies (Burnham, 1990). Peer review evolved to become a largely outsourced process, which still persists in modern scholarly publishing today, where publishers call upon external specialists to validate journal submissions. The current system of peer review only became more widespread in the mid 20th century (and in some disciplines, the late 20th century or early 21st; see Graf, 2014, for an example of a major philological journal which began systematic peer review in 2011). Nature, now considered a top journal, did not implement such a formal peer review process until 1967 (nature.com/nature/history/timeline_1960s.html).\n\nThis editor-led process of peer review became increasingly important in the post-World War II decades, due to the development of a modern academic prestige economy based on the perception of quality or excellence and symbolism surrounding journal-based publications (Baldwin, 2017a; Fyfe et al., 2017). The increasing professionalism of academies enabled commercial publishers to use peer review as a way of legitimizing their journals (Baldwin, 2015; Fyfe et al., 2017), and capitalized on the traditional perception of peer review as voluntary duty by academics to provide these services. A consequence of this was that peer review became a more homogenized process that enabled private publishing companies to establish a dominant, oligarchic marketplace position (Larivière et al., 2015). This represented a shift from peer review as a more synergistic activity between academics, to commercial entities selling it as an added value service back to the same academic community who was performing it freely for them. The estimated cost of peer review is a minimum of $1.9bn USD per year (in 2008; (Research Information Network, 2008)), representing a substantial vested financial interest in maintaining the current process of peer review (Smith, 2010). This figure does not even include the time spent by typically unpaid reviewers, or account for overhead costs in publisher management or the wasteful redundancy of the reject-resubmit cycle authors enter when chasing journal prestige (Jubb, 2016). The result of this is that peer review has now become enormously complicated. By allowing the process of peer review to become managed by a hyper-competitive industry, developments in scholarly publishing have become strongly coupled to the transforming nature of academic research institutes. These have evolved into internationally competitive businesses that strive for quality through publisher-mediated journals by attempting to align these products with the academic ideal of research excellence (Moore et al., 2017). Such a consequence is plausibly related to, or even a consequence of, broader shifts towards a more competitive neoliberal academia and society at large. Here, emphasis is largely placed on production and standing, value, or utility (Gupta, 2016), as opposed to the original primary focus of research on discovery and novel results.\n\n1.1.3 The peer review revolution. In the last several decades, there have been substantial efforts to decouple peer review from the publishing process (Figure 2; Schmidt & Görögh (2017)). This has typically been done either by adopting peer review as an overlay process on top of formally published research articles, or by pursuing a “publish first, filter later” protocol, with peer review taking place after the initial publication of research results ( McKiernan et al., 2016; Moed, 2007). Here, the meaning of “publication” becomes “making public”, as opposed to the traditional sense where it also implies peer reviewed. In fields such as Physics and Mathematics, it has traditionally been commonplace for authors to send their colleagues either paper or electronic copies of their manuscripts for pre-submission evaluation. Launched in 1991, arXiv (arxiv.org) formalized this process by creating a central network for whole communities to access such e-prints. Today, arXiv has more than one million e-prints from various research fields and receives more than 8,000 monthly submissions (arXiv, 2017). Here, e-prints or pre-prints are not formally peer reviewed prior to publication, but still undergo a certain degree of moderation in order to filter out non-scientific content. This practice represents a significant shift, as public dissemination was decoupled from a traditional peer review process, resulting in increased visibility and citation rates (Davis & Fromerth, 2007; Moed, 2007). The launch of Open Journal Systems (openjournalsystems.com; OJS) in 2001 offered a step towards bringing journals and peer review back to their community-led roots. As of 2015, the OJS platform provided the technical infrastructure and editorial and peer review workflow management support to more than 10,000 journals (Public Knowledge Project, 2016). Its exceptionally low cost was perhaps responsible for around half of these journals appearing in the developing world (Edgar & Willinsky, 2010).\n\nThe interactive data visualization is available at https://dgraziotin.shinyapps.io/peerreviewtimeline, and the source code and data are available at https://doi.org/10.6084/m9.figshare.5117260 (Graziotin, 2017).\n\nMore recently, there has been a new wave of innovation in peer review, which we term “the revolution” phase (Figure 2; note that this is a non-exhaustive overview of the peer review landscape). The pace of this is accelerating rapidly, with the majority of changes occurring in the last five to ten years. This could be related to initiatives such as the San Francisco Declaration on Research Assessment (ascb.org/dora/; DORA), that called for systemic changes in the way that scientific research outputs are evaluated. Digital-born journals, such as PLOS ONE, introduced commenting on published papers. This spurred developments in cross-publisher annotation platforms like PubPeer and PaperHive. Some journals, such as F1000 Research and The Winnower, rely exclusively on a model where peer review is conducted after the manuscripts are made publicly available. Other services, such as Publons, enable reviewers to claim recognition for their activities as referees. Platforms such as ScienceOpen provide a search engine combined with peer review across publishers on all documents, regardless of whether manuscripts have been previously reviewed. Each of these innovations has partial parallels to other social Web applications or platforms in terms of transparency, reputation, performance assessment, and community engagement. It remains to be seen whether these innovations and new models of evaluation will become more popular than traditional peer review.\n\nDue to the increasingly systematic use of external peer review, its processes have become entwined with the core activities of scholarly communication. Without approval through peer review to assess importance, validity, and journal suitability, research articles will not be sent to print. The historical motivation for selecting amongst submitted articles or distribution was primarily economic. With scholarly publishing turning into an essentially loss-making business, the costs of printing and paper needed to be limited (Fyfe, 2015). The rising number of submissions, particularly in the 20th century, required distributing the management of this selection process. While in the digital world the costs of dissemination have dropped, the marginal cost of publishing articles is far from zero (e.g., due to time and management, hosting, marketing, technical and ethical checks, among other services). The economic motivations for still imposing selectivity in a digital environment, and applying peer review as a mechanism for this, have received limited attention or questioning, and is often regarded as just how things are done. Selectivity is now often attributed to quality control, but is based on the false assumption that peer review requires careful selection of specific reviewers to assure a definitive level of adequate quality, termed the “Fallacy of Misplaced Focus” by Kelty et al. (2008).\n\nIn many cases, there is an attempt to link the goals of peer review processes with Mertonian norms (Lee et al., 2013; Merton, 1973) (i.e., universalism, communalism, disinterestedness, and organized skepticism) as a way of showing their relation to shared community values. The Mertonian norm of organized scepticism is the most obvious link, while the norm of disinterestedness can be linked to efforts to reduce systemic bias, and the norm of communalism to the expectation of contribution to peer review as part of community membership (i.e., duty). In contrast to the emphasis on supposedly shared social values, relatively little attention has been paid to the diversity of processes of peer review across journals, disciplines, and time. This is especially the case as the (scientific) scholarly community appears overall to have a strong investment in a “creation myth” that links the beginning of scholarly publishing—the founding of The Philosophical Transactions of the Royal Society—to the invention of peer review. The two are often regarded to be coupled by necessity, largely ignoring the complex and interwoven history of peer review and publishing. This has consequences, as the individual identity as a scholar is strongly tied to specific forms of publication that are evaluated in particular ways (Moore et al., 2017). A scholar’s first research article, PhD thesis, or first book are significant life events. Membership of a community, therefore, is validated by the peers who review this newly contributed work. Community investment in the idea that these processes have “always been followed” appears very strong, but ultimately remains a fallacy.\n\nAs mentioned above, there is an increasing quantity and quality of research that examines how publication processes, selection, and peer review evolved from the 17th to the early 20th century, and how this relates to broader social patterns (Baldwin, 2017a; Baldwin, 2017b; Moxham & Fyfe, 2016). However, there is much less research critically exploring the diversity of selection and peer review processes in the mid- to late-20th century. Indeed, there seems to be a remarkable discrepancy between the historical work we do have (Baldwin, 2017a; Gupta, 2016; Shuttleworth & Charnley, 2016) and apparent community views that “we have always done it this way,” alongside what sometimes feels like a wilful effort to ignore the current diversity of practice.\n\nSuch a discrepancy between a dynamic history and remembered consistency could be a consequence of peer review processes being central to both scholarly identity as a whole and to the identity and boundaries of specific communities (Moore et al., 2017). Indeed, this story linking identity to peer review is taught to junior researchers as a community norm, often without the much-needed historical context. More work on how peer review, alongside other community practices, contributes to community building and sustainability would be valuable. Examining criticisms of conventional peer review and proposals for change through the lens of community formation and identity may be a productive avenue for future research.\n\nIn spite of its clear relevance, widespread acceptance, and long-standing practice, the academic community does not appear to have a clear consensus on the operational functionality of peer review, and what its effects in a diverse modern research world are. There is a discrepancy between how peer review is regarded as a process, and how it is actually performed. While peer review is still generally perceived as key to quality control for research, others have begun to note that mistakes are becoming ever more frequent in the process (Margalida & Colomer, 2016; Smith, 2006), or at least that peer review is problematic and not being applied as rigorously as generally perceived (Cole, 2000; Eckberg, 1991; Ghosh et al., 2012; Jefferson et al., 2002; Kostoff (1995); Ross-Hellauer, 2017; Schroter et al., 2006; Walker & Rocha da Silva, 2015). One consequence of this is that COPE, the Committee on Publication Ethics (publicationethics.org), was established in 1997 to address potential cases of abuse and misconduct during the publication process. Yet, the effectiveness of this initiative at a system-level remains unclear. A popular editorial in The BMJ stated that peer review is “slow, expensive, profligate of academic time, highly subjective, prone to bias, easily abused, poor at detecting gross defects, and almost useless at detecting fraud,” with evidence supporting each of these quite serious allegations (Smith, 2006). However, beyond editorials, there now exists a substantial corpus of studies that critically examines the technical aspects of peer review. Taken together, this should be extremely worrisome, especially given that traditional peer review is still viewed almost dogmatically as a gold standard for the publication of research results, and as the process which mediates knowledge dissemination to the public.\n\nThe issue is that, ultimately, this uncertainty in standards and implementation can potentially lead to, or at least be viewed as the cause of, widespread failures in research quality and integrity (Ioannidis, 2005; Jefferson et al., 2002) and even the rise of formal retractions in extreme cases (Steen et al., 2013). Issues resulting from peer review failure range from simple gate-keeping errors, based on differences in opinion of the perceived impact of research, to failing to detect fraudulent or incorrect work, which then enters the scientific record (Baxt et al., 1998; Gøtzsche, 1989; Haug, 2015; Moore et al., 2017; Pocock et al., 1987; Schroter et al., 2004; Smith, 2006). A final issue regards peer review by and for non-native English speaking authors, which can lead to cases of linguistic inequality and language-oriented research segregation, in a world where research is increasingly becoming more globally competitive (Salager-Meyer, 2008; Salager-Meyer, 2014). All of this suggests that, while the idea of peer review remains logical, it is the implementation of it that requires attention.\n\n1.3.1 Peer review needs to be peer reviewed. Attempts to reproduce how peer review selects what is worthy of publication demonstrate that the process is generally adequate for detecting reliable research, but often fails to recognize the research that has the greatest impact (Mahoney, 1977; Moore et al., 2017; Siler et al., 2015). Many now regard the traditional peer review model as sub-optimal in that it causes publication delays, impacting the communication of novel research (Bornmann & Daniel, 2010; Brembs, 2015; Eisen, 2011; Jubb, 2016; Vines, 2015b). Reviewer fatigue (Breuning et al., 2015) and redundancy when articles go through multiple rounds of peer review at different journal venues (Moore et al., 2017; Jubb, 2016) are just some of the criticisms levied at the technical implementation of peer review. In addition, some view traditional peer review as flawed because it operates within a closed and opaque system. This makes it impossible to trace the discussions that led to (sometimes substantial) revisions to the original research (Bedeian, 2003), as well as the decision process leading to the final publication.\n\nOn top of all of these potential issues, some critics go even further in stating that, at its worst, peer review can be seen as detrimental to research. By operating as a closed system, it protects the status quo and suppresses research viewed as radical, innovative, or contrary to the theoretical perspectives of referees (Alvesson & Sandberg, 2014; Benda & Engels, 2011; Horrobin, 1990; Mahoney, 1977; Merton, 1968), even though it is precisely these factors that underpin and advance research. As a consequence, questions to the competency and integrity of traditional peer review arise, such as: who are the gatekeepers and how are their gates constructed; what is the balance between author-reviewer-editor tensions; what are the inherent biases associated with this; does this enable a fair or structurally inclined system of peer review to exist; and what are the repercussions for this on our knowledge generation and communication systems?\n\nIn spite of all of these criticisms, it remains clear that the ideal of peer review still plays a fundamental role in scholarly communication (Goodman et al., 1994; Pierie et al., 1996; Ware, 2008) and retains a high level of respect from the research community (Bedeian, 2003; Greaves et al., 2006; Gibson et al., 2008). One primary reason why peer review has persisted is that it remains a unique way of assigning credit and differentiating research publications from other types of literature, including blogs, media articles, and books. This perception, combined with a general lack of awareness or appreciation of the historic context of peer review, research examining its potential flaws, and the conflation of the process with the ideology, has sustained its ubiquitous usage and continued proliferation in academia. This has led to the widely-held perception that peer review is a singular and static process, and to its acceptance as a social norm. It is difficult to move away from a process that has now become so deeply embedded within oligarchic research institutes. The consequence of this is that, irrespective of any systemic flaws, peer review remains one of the essential pillars of trust when it comes to scientific communication (Haider & Åström, 2017).\n\nIn this article, we summarize the ebb and flow of the debate around the various and complex aspects of conventional (editorially-controlled) peer review. In particular, we highlight how innovative systems are attempting to resolve the major issues associated with traditional models, explore how new platforms could improve the process in the future, and consider what this means for the identity, role, and purpose of peer review within diverse research communities. The aim of this discussion is not to undermine any specific model of peer review in a quest for systemic upheaval, or to advocate any particular alternative model. Rather, we acknowledge that the idea of peer review is critical for research and advancing our knowledge, and as such we provide a foundation here for future exploration and creativity in diversifying and improving an essential component of scholarly communication.\n\n\n2 The traits and trends affecting modern peer review\n\nOver time, three principal forms of journal peer review have evolved: single blind, double blind, and open (Table 1). Of these, single blind, where reviewers are anonymous but authors are not, is the most widely-used in most disciplines because the process is comparably less onerous and less expensive to operate than the alternatives. double blind peer review, where both authors and reviewers are reciprocally anonymous, requires considerable effort to remove all traces of the author’s identity from the manuscript under review (Blank, 1991). For a detailed comparison of double versus single blind review, Snodgrass (2007) provides an excellent summary. These are generally considered to be the traditional forms of peer review, with the advent of open peer review introducing substantial additional complexity into the discussion (Ross-Hellauer, 2017).\n\nThe diversification of peer review is intrinsically coupled with wider developments in scholarly publishing. When it comes to the gate-keeping function of peer review, innovation is noticeable in some digital-only, or “born open,” journals, such as PLOS ONE and PeerJ. These explicitly request referees to ignore any notion of novelty, significance, or impact, before it becomes accessible to the research community. Instead, reviewers are asked to focus on whether the research was conducted properly and that the conclusions are based on the presented results. This arguably more objective method has met some resistance, even receiving the somewhat derogatory term “peer review lite” from some corners of the scholarly publishing industry (Pinfield, 2016). Such a perception is largely a hangover from the commercial age of publishing, and now seems superfluous and discordant with any modern Web-based model of scholarly communication. The relative timing of peer review to publication is a further major innovation, with journals such as F1000 Research publishing prior to any formal peer review process. Some of the advantages and disadvantages of these different variations of open peer review are explored in Table 2.\n\nNPRC: Neuroscience Peer Review Consortium.\n\nNovel ideas about “Open Peer Review” (OPR) systems are rapidly emerging, and innovation has been accelerating over the last several years (Figure 2; Table 3). The advent of OPR is complex, and often multiple aspects of peer review are used inter-changeably or are conflated without appropriate prior definition. Currently, there is no formally established definition of OPR that is accepted by the scholarly research and publishing community (Ford, 2013). The most simple definitions by McCormack (2009) and Mulligan et al. (2008) presented OPR as a process that does not attempt “to mask the identity of authors or reviewers” (McCormack, 2009, p.63), thereby explicitly referring to open in terms of personal identification or anonymity. Ware (2011, p.25) expanded on reviewer disclosure practices: “Open peer review can mean the opposite of double blind, in which authors’ and reviewers’ identities are both known to each other (and sometimes publicly disclosed), but discussion is complicated by the fact that it is also used to describe other approaches such as where the reviewers remain anonymous but their reports are published.” Other authors define OPR distinctly, for example by including the publication of all dialogue during the process (Shotton, 2012), or running it as a publicly participative commentary (Greaves et al., 2006). A recent survey by OpenAIRE found 122 different definitions of OPR in use, exemplifying the extent of this issue. This diversity was distilled into a single proposed definition comprising seven different open traits: participation, identity, reports, interaction, platforms, pre-review manuscripts, and final-version commenting (Ross-Hellauer, 2017).\n\nA core question is how to transform traditional peer review into a process aligned with the latest advances in what is now widely termed “open science”. This is tied to broader developments in how we as a society communicate, thanks to the inherent capacity that the Web provides for open, collaborative, and social communication. Many of the suggestions and new models for improving peer review are geared towards increasing the transparency and ultimately the reliability, efficiency, and accountability of the publishing process, and aligning peer review norms to support these aims. These traits are desired by all actors in the system, and increasing transparency moves peer review towards a more open model.\n\nHowever, the context of this transparency and the implications of different levels of transparency at different stages of the review process are both very rarely explored, and achieving transparency is difficult at a variety of levels. How and where we inject transparency into the system has implications for the magnitude of transformation and, therefore, the general concept of OPR is highly heterogeneous in meaning, scope, and consequences. New suggestions to modify peer review vary, between fairly incremental small-scale changes, to those that encompass an almost total and radical transformation of the present system. The various parts of the “revolutionary” phase of peer review undoubtedly have different combinations of these OPR traits, and within this remains a very heterogeneous landscape. Table 3 provides an overview of the advantages and disadvantages of the different approaches to anonymity and openness in peer review.\n\nIn this article, we regard OPR as a process fulfilling any of the following three primary criteria:\n\n1. Referee names are identified to the authors and the readership;\n\n2. Referee reports are made publicly available under an open license;\n\n3. Peer review is not restricted to invited referees only.\n\nWith all of these complex evolutionary trajectories, it is clear that peer review is undergoing a phase of experimentation in line with the evolving scholarly ecosystem. However, despite the range of new innovations, the engagement with these experimental open models is still far from common. The entrenchment of the ubiquitously practiced and much more favored traditional model (which, as noted above, is also diverse) is ironically non-traditional, but nonetheless currently revered. Practices such as self-publishing and predatory or deceptive publishing cast a shadow of doubt on the validity of research posted openly online that follow these models, including those with traditional scholarly imprints (Fitzpatrick, 2011a; Tennant et al., 2016). The inertia hindering widespread adoption of new models of peer review can be ascribed to what is often termed “cultural inertia” within scholarly research. Cultural inertia, the tendency of communities to cling to a traditional trajectory, is shaped by a complex ecosystem of individuals and groups. These often have highly polarized motivations (i.e., capitalistic commercialism versus knowledge generation versus careerism versus output measurement), and an academic hierarchy that imposes a power dynamic that can suppress innovative practices (Burris, 2004; Magee & Galinsky, 2008).\n\nThe ongoing discussions and innovations around peer review (and OPR) can be sorted into four main categories, which are examined in more detail below. Each of these feed into the wider issues of incentivizing engagement, providing appropriate recognition and certification, and quality control and moderation:\n\n1. How can referees receive credit or recognition for their work, and what form should this take;\n\n2. Should referee reports be published alongside manuscripts;\n\n3. Should referees remain anonymous or have their identities disclosed;\n\n4. Should peer review occur prior or subsequent to the publication process (i.e., publish then filter).\n\nA vast majority of researchers see peer review as an integral and fundamental part of their work. They often even consider peer review to be part of an altruistic cultural duty or a quid pro quo service, closely associated with the identity of being part of their research community. Generally, journals do not provide any remuneration or compensation for these services. Notable exceptions are the UK-based publisher Veruscript (veruscript.com/about/who-we-are) and Collabra (collabra.org/about/our-model), published by University of California Press. To be invited to review a research article is perceived as a great honor, especially for junior researchers, due to the recognition of expertise—i.e., the attainment of the level of a peer. However, the current system is facing new challenges as the number of published papers continues to increase rapidly (Albert et al., 2016), with more than one million articles published in peer reviewed, English-language journals every year (Larsen & Von Ins, 2010). Some estimates are even as high as 2–2.5 million per year (Plume & van Weijen, 2014), and this number is expected to double approximately every nine years at current rates (Bornmann & Mutz, 2015). There are several possible solutions to this issue:\n\nIncrease the total pool of potential referees,\n\nIncrease acceptance rates to avoid review duplication,\n\nDecrease the number of referees per paper, and/or\n\nDecrease the time spent on peer review.\n\nOf these, the latter two can both potentially reduce the quality of peer review, open or otherwise, and therefore affect the overall quality of published research. Paradoxically, as the Internet empowers us to communicate information virtually instantaneously, the turn around time for peer reviewed publications is as far from this as it ever has been. One potential solution to this is to encourage referees by providing additional recognition and credit for their work. The present lack of bona fide incentives for referees is perhaps the main factor responsible for indifference to editorial outcomes, which ultimately leads to the increased proliferation of low quality research (D’Andrea & O’Dwyer, 2017).\n\n2.2.1 Traditional methods of recognition. One current way to recognize peer review is to thank anonymous referees in the Acknowledgement sections of published papers. In these cases, the referees will not receive any public recognition for their work, unless they explicitly agree to sign their reviews. Another common form of acknowledgement is a private thank you note from the journal or editor, which usually takes the form of an automated email upon completion of the review. In addition, journals often list and thank all reviewers in a special issue or on their website once a year, thus providing another way to credit reviewers. Another idea that journals and publishers have tried implementing is to list the best reviewers for their journal (e.g., by Vines (2015a) for Molecular Ecology), or, on the basis of a suggestion by Pullum (1984), naming referees who recommend acceptance in the article colophon (a single blind version of this recommendation was adopted by Digital Medievalist from 2005–2016; see Wikipedia contributors, 2017, and bit.ly/DigitalMedievalistArchive for examples preserved in the Internet Archive). Digital Medievalist stopped using this model and removed the colophon as part of its move to the Open Library of Humanities; cf. journal.digitalmedievalist.org). As such, authors can then integrate this into their scholarly profiles in order to differentiate themselves from other researchers or referees. Currently, most tenure and review committees do not consider peer review activities as required or sufficient in the process of professional advancement or tenure evaluation. Instead, it is viewed as expected or normal behaviour for all researchers to contribute in some form to peer review.\n\n2.2.2 Increasing demand for recognition. Traditional approaches of credit fall short of any sort of systematic feedback or recognition, such as that granted through publications. A change here is clearly required for the wealth of currently unrewarded time and effort given to peer review by academics. A recent survey of nearly 3,000 peer reviewers by the large commercial publisher Wiley showed that feedback and acknowledgement for work as referees are valued far above either cash reimbursements or payment in kind (Warne, 2016). As of today, peer review is poorly acknowledged by practically all research assessment bodies, institutions, granting agencies, as well as publishers. Wiley’s survey reports that 80% of researchers agree that there is insufficient recognition for peer review as a valuable research activity and that researchers would actually commit more time to peer review if it became a formally recognized activity for assessments, funding opportunities, and promotion (Warne, 2016). While this may be true, it is important to note that commercial publishers, including Wiley, have a vested interest in retaining the current, freely provided service of peer review since this is what provides their journals the main stamp of legitimacy and quality (“added value”) as society-led journals. Therefore, one of the root causes for the lack of appropriate recognition and incentivization is, ironically, publishers themselves, who have strong motivations to find non-monetary forms of reviewer recognition. Indeed, the business model of almost every large scholarly publisher is predicated on free work by peer reviewers, and it is unlikely that the present system would function financially with market-rate reimbursement of peer reviewers. Hence, this survey could represent a biased view of the actual situation. Other research shows a similar picture, with approximately 70% of respondents to a small survey done by Nicholson & Alperin (2016) indicating that they would list peer review as a professional service on their curriculum vitae. 27% of respondents mentioned formal recognition in assessment as a factor that would motivate them to participate in public peer review. These numbers indicate that the lack of credit referees receive for peer review is a contributing factor to the perceived stagnation of the traditional models. Furthermore, acceptance rates are lower in humanities and social sciences, and higher in physical sciences and engineering journals (Ware, 2008). This means there are distinct disciplinary variations in the number of reviews performed by a researcher relative to their publications, and suggests that there is scope for using this to either provide different incentive structures or to increase acceptance rates and therefore decrease referee fatigue (Lyman, 2013).\n\n2.2.3 Progress in crediting peer review. Any acknowledgement model to credit reviewers also raises the obvious question of how to facilitate this model within an anonymous peer review system. By incentivizing peer review, much of its potential burden can be alleviated by widening the potential referee pool. This can also help to diversify the process and inject transparency into peer review, a solution that is especially appealing when considering that it is often a small minority of researchers who perform the vast majority of peer reviews (Fox et al., 2017; Gropp et al., 2017); for example, in biomedical research, only 20 percent of researchers perform 70–95 percent of the reviews (Kovanis et al., 2016). In 2014, a working group on peer review services (CASRAI) was established to “develop recommendations for data fields, descriptors, persistence, resolution, and citation, and describe options for linking peer-review activities with a person identifier such as ORCID” (Paglione & Lawrence, 2015). The idea here is that by being able to standardize peer review activities, it becomes easier to describe, attribute, and therefore recognize and reward them.\n\nThe Publons platform provides a semi-automated mechanism to formally recognize the role of editors and referees who can receive due credit for their work as referees, both pre- and post-publication. Researchers can also choose if they want to publish their full reports depending on publisher and journal policies. Publons also provides a ranking for the quality of the reviewed research article, and users can endorse, follow, and recommend reviews. Other platforms, such as F1000 Research and ScienceOpen, link post-publication peer review activities with CrossRef DOIs to make them more citable, essentially treating them equivalent to a normal Open Access research paper. ORCID (Open Researcher and Contributor ID) provides a stable means of integrating with platforms such as Publons and ImpactStory in order to receive due credit for reviews. ORCID is rapidly becoming part of the critical infrastructure for OPR, and greater shifts towards open scholarship (Dappert et al., 2017). Exposing peer reviews through these platforms links accountability to receiving credit. Therefore, they offer possible solutions to the dual issues of rigor and reward, while potentially ameliorating the growing threat of reviewer fatigue. Whether such initiatives will be successful remains to be seen, although Publons was recently acquired by Clarivate Analytics, suggesting that the process could become commercialized as this domain rapidly evolves (Van Noorden, 2017). In spite of this, the outcome is most likely to be dependent on whether funding agencies and those in charge of tenure, hiring, and promotion will use peer review activities to help evaluate candidates. This is likely dependent on whether research communities themselves choose to embrace any such crediting or accounting systems for peer review.\n\nThe rationale behind publishing referee reports lies in providing increased context and transparency to the peer review process—the making of the sausage, so to speak. Often, valuable insights are shared in reviews that would otherwise remain hidden if not published. By publishing reports, peer review then has the potential to become a supportive and collaborative process that is viewed more as an ongoing dialogue between groups of scientists to progressively assess the quality of research. Furthermore, the reviews themselves are opened up for analysis and inspection, including how authors respond to reviews themselves, which adds an additional layer of quality control and a means for accountability and verification. There are additional educational benefits to publishing peer reviews, such as training purposes or for journal clubs. At the present, some publisher policies are extremely vague about the re-use rights and ownership of peer review reports (Schiermeier, 2017).\n\nIn a study of two journals, one where reports were not published and another where they were, Bornmann et al. (2012) found that publicized comments were much longer. Furthermore, there was an increased chance that they may result in a constructive dialogue between the author, reviewers, and wider community, and might therefore be better for improving the content of a manuscript. On the other hand, unpublished reviews tend to have had more of a selective function to determine whether a manuscript is appropriate for a particular journal (i.e., focusing on the editorial process). Therefore, depending on the journal, different types of peer review could be better suited to perform different functions, and therefore optimized in that direction. Transparency of the peer review process can also be used as an indicator for peer review quality, thereby potentially enabling the tool to predict quality in new journals in which the peer review model is known (Godlee, 2002; Morrison, 2006; Wicherts, 2016), if desired. Journals with higher transparency ratings were less likely to accept flawed papers and showed a higher impact as measured by Google Scholar’s h5-index.\n\nIt is ironic that, while assessments of articles can never be evidence-based without the publication of referee reports, they are still almost ubiquitously regarded as having an authoritative stamp of quality. The issue here is that the attainment of peer reviewed status will always be based on an undefined, and only ever relative, quality threshold due to the opacity of the process. This is quite an unscientific practice, and instead, researchers rely almost entirely on heuristics and trust for a concealed process and the intrinsic reputation of the journal, rather than anything legitimate. This can ultimately result in what is termed the “Fallacy of Misplaced Finality”, described by Kelty et al. (2008), as the assumption that research has a single, final form, to which everyone applies different criteria of quality.\n\nPublishing peer review reports appears to have little or no impact on the overall process but may encourage more civility from referees. In a small survey, Nicholson & Alperin (2016) found that approximately 75% of survey respondents (n=79) perceived that public peer review would change the tone or content of the reviews, and 80% of responses indicated that performing peer reviews that would be eventually be publicized would not require a significantly higher amount of work. However, the responses also indicated that an incentive is needed for referees to engage in open peer review. This would include recognition by performance review or tenure committees (27%), peers publishing their reviews (26%), being paid in some way such as with an honorarium or waived APC (24%), and getting positive feedback on reviews from journal editors (16%). Only 3% (one response) indicated that nothing could motivate them to participate in an open peer review of this kind. Leek et al. (2011) showed that when referees’ comments were made public, significantly more cooperative interactions were formed, while the risk of incorrect comments decreased. Moreover, referees and authors who participated in cooperative interactions had a reviewing accuracy rate that was 11% higher. On the other hand, the possibility of publishing the reviews online has also been associated with a high decline rate among potential peer reviewers, and an increase in the amount of time taken to write a review, but with no effect on review quality (van Rooyen et al., 2010). This suggests that the barriers to publishing review reports are inherently social, rather than technical.\n\nWhen BioMed Central launched in 2000, it quickly recognized the value in including both the reviewers’ names and the peer review history (pre-publication) alongside published manuscripts in their medical journals. Since then, further reflections on open peer review (Godlee, 2002) led to the adoption of a variety of OPR models. For example, the Frontiers series now publishes all referee names alongside articles, EMBO journals publish a review process file with the articles, with referees remaining anonymous but editors being named, and PLOS added public commenting features to articles they published in 2009. More recently, launched journals such as PeerJ have a system where both the reviews and the names of the referees can optionally be made public, and journals such as Nature Communications and the European Journal of Neuroscience have started to adopt this method of OPR as well.\n\nUnresolved issues with posting review reports include whether or not it should be conducted for ultimately unpublished manuscripts, the impact of author identification or anonymity, and if the announcement of author’s career stage has potential consequences on their reputations. Furthermore, the actual readership and usage of published reports remains ambiguous in a world where researchers are typically already inundated with published articles to read. The benefits of publicizing reports might not be seen until further down the line from the initial publication and, therefore, their immediate value might be difficult to convey and measure in current research environments. Finally, different populations of reviewers with different cultural norms and identities will undoubtedly have varying perspectives on this issue, and it is unlikely that any single policy or solution to posting referee reports will ever be widely adopted.\n\nThere are different levels of bi-directional anonymity throughout the peer review process, including whether or not the referees know who the authors are but not vice versa (single blind, the most common; (Ware, 2008)), or whether both parties remain anonymous to each other (double blind) (Table 1). Traditional double blind review is based on the idea that peer evaluations should be impartial and based on the research, not ad hominem, but there has been considerable discussion over whether reviewer identities should remain anonymous (e.g., Baggs et al. (2008); Pontille & Torny (2014); Snodgrass (2007)) (Figure 3). Models such as triple-blind peer review even go a step further, where authors and their affiliations are reciprocally anonymous to the handling editor and the reviewers. This attempts to nullify the effects of one’s scientific reputation, institution, or location on the peer review process, and is employed at the Open Access journal Science Matters (sciencematters.io), launched in early 2016.\n\nStrong, but often conflicting arguments and attitudes exist for both sides of the anonymity debate (see e.g., Prechelt et al. (2017)). In theory, anonymous reviewers are protected from potential backlashes for expressing themselves fully and therefore are more likely to be more honest in their assessments. Further, there is some evidence to suggest that double blind review can increase the acceptance rate of women-authored articles in the published literature (Darling, 2015). However, this kind of anonymity can be difficult to protect, as there are ways in which identities can be revealed, albeit non-maliciously, such as through language and phrasing, prior knowledge of the research and a specific angle being taken, previous presentation at a conference, or even simple Web-based searches.\n\nWhile there is much potential value in anonymity, the corollary is also problematic in that anonymity can lead to reviewers being more aggressive, biased, negligent, orthodox, entitled, and politicized in their language and evaluation, as they have no fear of negative consequences for their actions other than from the editor. (Lee et al., 2013; Weicher, 2008). Furthermore, by protecting the referees’ identities journals lose an aspect of the prestige, quality, and validation in the review process, leaving researchers to guess or assume this important aspect postpublication. The transparency associated with signed peer review aims to avoid competition and conflicts of interest that can potentially arise due to the fact that referees are often the closest competitors to the authors, as they will naturally tend to be the most competent to assess the research (Campanario, 1998a; Campanario, 1998b). Eponymous peer review has the potential to encourage increased civility, accountability, and more thoughtful reviews (Boldt, 2011; Cope & Kalantzis, 2009; Fitzpatrick, 2010; Janowicz & Hitzler, 2012; Lipworth et al., 2011; Mulligan et al., 2013), as well as extending the process to become more of an ongoing, community-driven dialogue rather than a singular, static event (Bornmann et al., 2012; Maharg & Duncan, 2007). However, there is scope for the peer review to become less critical, skewed, and biased by community selectivity. If the anonymity of the reviewers is removed while maintaining author anonymity at any time during peer review, a skew and extreme accountability is imposed upon the reviewers, while authors remain relatively protected from any potential prejudices against them. However, such transparency provides, in theory, a mode of validation and should mitigate corruption as any association between authors and reviewers would be exposed. Yet, this approach has a clear disadvantage, in that accountability becomes extremely one-sided. Another possible result of this is that reviewers could be stricter in their appraisals within an already conservative environment, and thereby further prevent the publication of research.\n\n2.4.1 Reviewing the evidence. Baggs et al. (2008) investigated the beliefs and preferences of reviewers about blinding. Their results showed double blinding was preferred by 94% of reviewers, although some identified advantages to an un-blinded process. When author names were blinded, 62% of reviewers could not identify the authors, while 17% could identify authors ≤10% of the time. Walsh et al. (2000) conducted a survey in which 76% of reviewers agreed to sign their reviews. In this case, signed reviews were of higher quality, were more courteous, and took longer to complete than unsigned reviews. Reviewers who signed were also more likely to recommend publication. In their study to explore the review process from the reviewers’ perspectives, Snell & Spencer (2005) found that reviewers would be willing to sign their reviews and feel that the process should be transparent. Yet, a similar study by Melero & Lopez-Santovena (2001) found that 75% of surveyed respondents were in favor of reviewer anonymity, while only 17% were against it.\n\nA randomized trial showed that blinding reviewers to the identity of authors improved the quality of the reviews (McNutt et al., 1990). This trial was repeated on a larger scale by Justice et al. (1998) and Van Rooyen et al. (1999), with neither study finding that blinding reviewers improved the quality of reviews. These studies also showed that blinding is difficult in practice as many manuscripts include clues on authorship. Jadad et al. (1996) analyzed the quality of reports of randomized clinical trials and concluded that blind assessments produced significantly lower and more consistent scores than open assessments. The majority of additional evidence suggests that anonymity has little impact on the quality or speed of the review or of acceptance rates (Isenberg et al., 2009; Justice et al., 1998; van Rooyen et al., 1998), but revealing the identity of reviewers may lower the likelihood that someone will accept an invitation to review (Van Rooyen et al., 1999). Revealing the identity of the reviewer to a co-reviewer also has a small, editorially insignificant, but statistically significant beneficial effect on the quality of the review (van Rooyen et al., 1998). Authors who are aware of the identity of their reviewers may also be less upset by hostile and discourteous comments (McNutt et al., 1990). Other research found that signed reviews were more polite in tone, of higher quality, and more likely to ultimately recommend acceptance (Walsh et al., 2000).\n\n2.4.2 The dark side of identification. The debate of signed versus unsigned reviews is not to be taken lightly. Early career researchers in particular are some of the most conservative in this area as they may be afraid that by signing overly critical reviews (i.e., those which investigate the research more thoroughly), they will become targets for retaliatory backlashes from more senior researchers. In this case, the justification for reviewer anonymity is to protect junior researchers, as well as other marginalized demographics, from bad behaviour. Furthermore, author anonymity could potentially save junior authors from public humiliation from more established members of the research community, should they make errors in their evaluations. These potential issues are at least a part of the cause towards a general attitude of conservatism from the research community towards OPR. Indeed, they come up as the most prominent resistance factor in almost every formal discussion on the top of open peer review (e.g., Darling (2015); Godlee et al. (1998); McCormack (2009); Pontille & Torny (2014); Snodgrass (2007)van Rooyen et al. (1998)). However, it is not immediately clear how this widely-exclaimed but poorly documented potential abuse of signed-reviews is any different from what would occur in a closed system anyway, as anonymity provides a potential mechanism for referee abuse. The fear that most backlashes would be external to the peer review itself, and indeed occur in private, is probably the main reason why such abuse has not been widely documented. However, it can also be argued that by reviewing with the prior knowledge of open identification, such backlashes are prevented since researchers do not want to tarnish their reputations in a public forum. Under these circumstances, openness becomes a means to hold both referees and authors accountable for their public discourse, as well as making the editors’ decisions on referee and publishing choice public. Either way, there is little documented evidence that such retaliations actually occur either commonly or systematically. If they did, then publishers that employ this model such as Frontiers or BioMed Central would be under serious question, instead of thriving as they are.\n\nIn an ideal world, we would expect that strong, honest, and constructive feedback is well received by authors, no matter their career stage. Yet, it seems that this is not the case, or at least there seems to be the very real perception that it is not, and this is just as important from a social perspective. Retaliations to referees in such a negative manner represent serious cases of academic misconduct (Fox, 1994; Rennie, 2003). It is important to note, however, that this is not a direct consequence of OPR, but instead a failure of the general academic system to mitigate and act against inappropriate behavior. Increased transparency can only aid in preventing and tackling the potential issues of abuse and publication misconduct, something which is almost entirely absent within a closed system. COPE provides advice to editors and publishers on publication ethics, and on how to handle cases of research and publication misconduct, including during peer review. COPE could be used as the basis for developing formal mechanisms adapted to innovative models of peer review, including those outlined in this paper. Any new OPR ecosystem could also draw on the experience accumulated by Online Dispute Resolution (ODR) researchers and practitioners over the past 20 years. ODR can be defined as “the application of information and communications technology to the prevention, management, and resolution of disputes” (Katsh & Rule, 2015), and could be implemented to prevent, mitigate, and deal with any potential misconduct during peer review alongside COPE. Therefore, the perceived danger of author backlash is highly unlikely to be acceptable in the current academic system, and if it does occur, it can be dealt with through increased transparency. Furthermore, bias and retaliation exist even in a double blind review process (Baggs et al., 2008; Snodgrass, 2007; Tomkins et al., 2017), which is generally considered to be more conservative or protective. Such widespread identification of bias highlights this as a more general issue within peer review and academia more broadly, and we should be careful not to attribute it to any particular mode or trait of peer review. This is particularly relevant for more specialized fields, where the pool of potential authors and reviewers is relatively small (Riggs, 1995). Nonetheless, careful engagement with researchers, especially high-risk or marginalized communities, should be a necessary and vital step prior to implementation of any system of reviewer transparency.\n\n2.4.3 The impact of identification and anonymity on bias. One of the biggest criticisms levied at peer review is that, like many human endeavours, it is intrinsically biased and not the objective and impartial process many regard it to be. The question is no longer about whether or not it is biased, but to what extent it is in different social dimensions. One of the major issues is that peer review suffers from systemic confirmatory bias, with only results that are deemed as significant, statistically or otherwise, being selected for publication (Mahoney, 1977). This causes a distinct bias within the published research record (van Assen et al., 2014), as a consequence of perverting the research process itself by creating an incentive system that is almost entirely publication-oriented. Others have described the issues with such an asymmetric evaluation criteria as lacking the core values of a scientific process (Bon et al., 2017).\n\nThe evidence on whether there is bias in peer review against certain author demographics is mixed, but overwhelmingly in favor of systemic bias against women in article publishing (Budden et al., 2008; Darling, 2015; Grivell, 2006; Helmer et al., 2017; Lerback & Hanson, 2017; Lloyd, 1990; McKiernan, 2003; Roberts & Verhoef, 2016; Smith, 2006; Tregenza, 2002) (although see Blank (1991); Webb et al. (2008); Whittaker (2008)). After the journal Behavioural Ecology adopted double blind peer review in 2001, there was a significant increase in accepted manuscripts by women first authors; an effect not observed in similar journals that did not change their peer review policy (Budden et al., 2008). One of the most recent public examples of this bias is the case where a reviewer told the authors that they should add more male authors to their study (Bernstein, 2015). More recently, it has been shown in the Frontiers journal series that women are under-represented in peer-review and that editors of both genders operate with substantial same-gender preference (Helmer et al., 2017). The most famous piece of evidence on bias against authors comes from a study by Peters & Ceci (1982) using psychology journals. They took 12 studies that came from prestigious institutions that had already been published in psychology journals. They retyped the papers, made minor changes to the titles, abstracts, and introductions but changed the authors’ names and institutions. The papers were then resubmitted to the journals that had first published them. In only three cases did the journals realize that they had already published the paper, and eight of the remaining nine were rejected—not because of lack of originality but because of the perception of poor quality. Peters & Ceci (1982) concluded that this was evidence of bias against authors from less prestigious institutions, although the deeper causes of this bias remain unclear at the present. A similar effect was found in an orthopaedic journal by Okike et al. (2016), where reviewers were more likely to recommend acceptance when the authors’ names and institutions were visible than when they were redacted. Further studies have shown that peer review is substantially positively biased towards authors from top institutions (Ross et al., 2006; Tomkins et al., 2017), due to the perception of prestige of those institutions and, consequently, of the authors as well. Further biases based on nationality and language have also been shown to exist (Dall’Aglio, 2006; Ernst & Kienbacher, 1991; Link, 1998; Ross et al., 2006; Tregenza, 2002).\n\nWhile there are relatively few large-scale investigations of the extent and mode of bias within peer review (although see Lee et al. (2013) for an excellent overview of the different levels in which bias can be potentially injected into the process), these studies together indicate that inherent biases are systemically embedded within the process, and must be accounted for prior to any further developments in peer review. This range of population-level investigations into attitudes and applications of anonymity, and the extent of any biases resulting from this, exposes a highly complex picture, and there is little consensus on its impact at a system-wide scale. However, based on these often polarised studies, it is inescapable to conclude that peer review is highly subjective, rarely impartial, and definitely not as homogeneous as it is often regarded.\n\nApplying a single, blanket policy regarding anonymity would greatly degrade the ability of science to move forward, especially without the flexibility to manage exceptions. The reasons to avoid one definite policy are the inherent complexity of peer review systems, the interplay with different cultural aspects within the various sub-sectors of research, and the difficulty in identifying whether anonymous or identified works are objectively better. As a general overview of the current peer review ecosystem, Nobarany & Booth (2017) recently recommended that, due to this inherent diversity, peer review policies and support systems should remain flexible and customizable to suit the needs of different research communities. We expect that, by emphasizing the different shared values across research communities, as well as their commonalities, we will see a new diversity of OPR processes developed across disciplines in the future. Remaining ignorant of this diversity of practices and inherent biases in peer review, as both social and physical processes, would be an unwise approach for future innovations.\n\nOne proposal to transform scholarly publishing is to decouple the concept of the journal and its functions (e.g., archiving, registration and dissemination) from peer review and the certification that this provides. Some even hail this decoupling process as the “paradigm shift” that scholarly publishing needs (Priem & Hemminger, 2012). Some publishers, journals, and platforms are now taking a more adventurous exploration of peer review that occurs subsequently to publication (Figure 3). Here, the principle is that all research deserves the opportunity to be published (usually pending some form of initial editorial selectivity), and that filtering through peer review occurs subsequently to the actual communication of research articles (i.e., a publish then filter process). This is often termed “post-publication peer review”, a confusing terminology based on what constitutes “publication” in the digital age, depending on whether it occurs on manuscripts that have been previously peer reviewed or not (blogs.openaire.eu/?p=1205). Numerous venues now provide inbuilt systems for post-publication peer review, including RIO, PubPub, ScienceOpen, The Winnower, and F1000 Research. In addition to the systems adopted by journals, other post-publication annotation and commenting services exist independent of any specific journal or publisher and operating across platforms, such as hypothes.is, PaperHive, and PubPeer.\n\nThe dotted border lines in the figure highlight this element, with boxes colored in orange representing decoupled steps from the traditional publishing model (0) and the ones colored gray depicting the traditional publishing model itself. Pre-submission peer review based decoupling (1) offers a route to enhance a manuscript before submitting it to a traditional journal; post-publication peer review based decoupling follows pre-print first mode through four different ways (2, 3, 4, and 5) for revision and acceptance. Dual-decoupling (3) is when a manuscript initially posted as a pre-print (first decoupling) is sent for external peer review (second decoupling) before its formal submission to a traditional journal. The asterisks in the figure indicate when the manuscript first enters the public view irrespective of its peer review status.\n\nInitiatives such as the Peerage of Science (peerageofscience.org), RUBRIQ (rubriq.com), and Axios Review (axiosreview.org; closed in 2017) have implemented a decoupled model of peer review. These tools work based on the same core principles as traditional peer review, but authors submit their manuscripts to the platforms first instead of journals. The platforms provide the referees, either via subject-specific editors or via self-managed agreements. After the referees have provided their comments and the manuscript has been improved, the platform forwards the manuscript and the referee reports to a journal. Some journal policies accept the platform reviews as if the reviews were coming from the journal’s pool of reviewers, while others still require the journal’s handling editor to look for additional reviewers. While these systems usually cost money for authors, these costs can sometimes be deducted from any publication fees once the article has been published. Journals accept deduction of these costs because they benefit by receiving manuscripts that have already been assessed for journal fit and have been through a round of revisions, thereby reducing their workload. A consortium of publishers and commercial vendors recently established the Manuscript Exchange Common Approach (MECA; manuscriptexchange.org) as a form of portable review in order to cut down inefficiency and redundancy. Yet, it still is in too early a stage to comment on its viability.\n\nLIBRE (openscholar.org.uk/libre) is a free, multidisciplinary, digital article repository for formal publication and community-based evaluation. Reviewers’ assessments, citation indices, community ratings, and usage statistics, are used by LIBRE to calculate multiparametric performance metrics. At any time, authors can upload an improved version of their article or decide to send it to an academic journal. Launched in 2013, LIBRE was subsequently combined with the Self-Journal of Science (sjscience.org) under the combined heading of Open Scholar (openscholar.org.uk). One of the tools that Open Scholar offers is a peer review module for integration with institutional repositories, which is designed to bring research evaluation back into the hands of research communities themselves (openscholar.org.uk/open-peer-review-module-for-repositories). Academic Karma is another new service that facilitates peer review of pre-prints from a range of sources (academickarma.org/).\n\n2.5.1 Pre-prints and overlay journals. In fields such as mathematics, astrophysics, or cosmology, research communities already commonly publish their work on arXiv (Larivière et al., 2014). To date, this platform has accumulated more than one million research documents – pre-prints or e-prints – and currently receives 8000 submissions a month with no costs to authors. arXiv also sparked innovation for a number of communication and validation tools within restricted communities, although these seem to be largely local, non-interoperable, and do not appear to have disrupted the traditional scholarly publishing process to any great extent (Marra, 2017). In other fields, the uptake of pre-prints has been relatively slower, although it is gaining momentum with the development of platforms such as bioRxiv and several newly established ones through the Center for Open Science, including engrXiv (engrXiv.org) and psyarXiv (psyarxiv.com), and social movements such as ASAPBio (asapbio.org). Manuscripts submitted to these pre-print servers are typically a draft version prior to formal submission to a journal for peer review. Primary motivation for this is the lengthy time taken for peer review and formal publication, and causes the timing of peer review to occur subsequent to making manuscripts public. However, sometimes these articles are not submitted anywhere else and form what some regard as grey literature (Luzi, 2000). Papers on digital repositories are cited on a daily basis and much research builds upon them, although they may suffer from a stigma of not having the scientific stamp of approval of peer review (Adam, 2010). Some journal policies explicitly attempt to limit their citation in peer-reviewed publications (e.g., Nature nature.com/nature/authors/gta/#a5.4 and Cell cell.com/cell/authors), and recently the scholarly publishing sector even attempted to discredit their recognition as valuable publications (asapbio.org/faseb). In spite of this, the popularity and success of pre-prints is testified by their citation records, with four of the top five venues in physics and maths being arXiv sub-sections (scholar.google.com/citations?view_op=top_venues&hl=en&vq=phy). Similarly, the single most highly cited venue in economics is the NBER Working Papers server (scholar.google.com/citations?view_op=top_venues&hl=en&vq=bus_economics), according to the Google Scholar h5-index.\n\nThe overlay journal, first described by Ginsparg (1997), is a novel type of journal that operates by having peer review as an additional layer on top of pre-prints. These have built on the concept of deconstructed journals (Smith, 1999), which decouple peer-review from publishing (Hettyey et al., 2012; Patel, 2014; Stemmle & Collier, 2013; Vines, 2015b). New overlay journals such as The Open Journal (theoj.org) or Discrete Analysis (discreteanalysisjournal.com) are exclusively peer review platforms that circumvent traditional publishing by utilizing the pre-existing infrastructure and content of pre-print servers like arXiv. Peer review is performed easily, rapidly, and cheaply, after initial publication of the articles. The reason they are termed “overlay” journals is that the articles remain on arXiv in their peer reviewed state, with the “journals” mostly comprising a simple list of links to these versions (Gibney, 2016).\n\nA similar approach to that of overlay journals is being developed by PubPub (pubpub.org), which allows authors to self-publish their work. PubPub then provides a mechanism for creating overlay journals that can draw from and curate the content hosted on the platform itself. This model incorporates the pre-print server and final article publishing into one contained system. EPISCIENCES is another platform that facilitates the creation of peer reviewed journals, with their content hosted on digital repositories (Berthaud et al., 2014). ScienceOpen provides editorially-managed collections of articles drawn from pre-prints and a combination of open access and non-open venues (e.g., scienceopen.com/collection/Science20). Editors compile articles to form a collection, write an editorial, and can invite referees to peer review the articles. This process is mediated by ORCID for quality control, and CrossRef and Creative Commons licensing for appropriate recognition. They are essentially equivalent to community-mediated overlay journals, but with the difference that they also draw on additional sources beyond pre-prints.\n\n2.5.2 Two-stage peer review and Registered Reports. Registered Reports represent a significant departure from conventional peer review in terms of relative timing and increased rigour (Chambers et al., 2014; Chambers et al., 2017; Nosek & Lakens, 2014). Here, peer review is split into two stages. Research questions and methodology (i.e., the study design itself) are subject to a first round of evaluation prior to any data collection or analysis taking place (Figure 4). If a protocol is found to be of sufficient quality to pass this stage, the study is then provisionally accepted for publication. Once the research has been completed and written-up, completed manuscripts are then subject to a second-stage of peer review which, in addition to affirming the soundness of the results, also confirms that data collection and analysis occurred in accordance with the originally described methodology. The format, originally introduced by the psychology journals Cortex and Perspectives in Psychological Science in 2013, is now used in some form by more than 40 journals (Nature Human Behaviour, 2017). Registered Reports are designed to boost research integrity by ensuring the publication of all research results, which helps reduce publication bias. As opposed to the traditional model of publication, where “positive” results are more likely to be published, results remain unknown at the time of review and therefore even “negative” results are equally as likely to be published. Such a process is designed to incentivize data-sharing, guard against dubious practices such as selective reporting of results (via so-called “p-hacking” and “HARKing”— Hypothesizing After the Results are Known) and low statistical power, and also prioritizes accurate reporting over that which is perceived to be of higher impact or publisher worthiness.\n\nEach peer review stage also includes editorial input.\n\n2.5.3 Peer Review by Endorsement. A relatively new mode of named pre-publication review is that of pre-arranged and invited review, originally proposed as author-guided peer review (Perakakis et al., 2010), which ScienceOpen terms Peer Review by Endorsement (PRE) (about.scienceopen.com/peerreview-by-endorsement-pre/). This has also been implemented at RIO, and is functionally similar to the Contributed Submissions of PNAS (pnas.org/site/authors/editorialpolicies.xhtml#contributed). This model requires an author to solicit reviews from their peers prior to submission in order to assess the suitability of a manuscript for publication. While some might see this as a potential bias, it is worth bearing in mind that many journals already ask authors who they want to review their papers, or who they should exclude. To avoid potential pre-submission bias, reviewer identities and their endorsements are made publicly available alongside manuscripts, which also removes any possible deleterious editorial criteria from inhibiting the publication of research. Also, PRE is much cheaper, legitimate, unbiased, faster, and more efficient alternative to the traditional publisher-mediated method. In theory, depending on the state of the manuscript, this means that submissions can be published much more rapidly, as less processing is required. PRE also has the potential advantage of being more useful to non-native English speaking authors by allowing them to work with editors and reviewers in their first languages.\n\nEndorsements and recommendations are a form of peer review that can facilitate re-use of published works. This has been most evident in the Open Educational Resources (OER) movement, in which peer review and testimonials on Open Education repositories, such as Merlot, form a way to filter the many resources available. Peer review, including recommendations, has been effectively utilized in the creation and sharing of Open Textbooks. Petrides et al. (2011) and Harley et al. (2010) found that proof of peer review by trusted experts was a significant factor leading to adoption of textbooks by instructors who expressed concern about the quality of a free textbook. Some OER reviewers are even paid for their reviews (Open Access Textbook Task Force, 2010), while other reviews are done by volunteer editors and the users of the resources. (info.merlot.org/merlothelp/merlot_peer_review_information.htm).\n\n2.5.4 Limitations of decoupled peer review. Despite a general appeal for post-publication peer review and considerable innovation in this field, the appetite among researchers is limited, reflecting an overall lack of engagement with the process (e.g., Nature (2010)). As recently as 2012, it was reported that relatively few platforms allowed users to evaluate manuscripts post-publication (Yarkoni, 2012). Even platforms such as PLOS have a restricted scope and limited user base: analysis of publicly available usage statistics indicate that at the time of writing, PLOS articles have each received an average of 0.06 ratings and 0.15 comments (see also Ware (2011)). Part of this may be due to how post-publication peer review is perceived culturally, with the name itself being anathema and considered an oxymoron, as most researchers usually consider a published article to be one that has already undergone formal peer review. At the present, it is clear that while there are numerous platforms providing decoupled peer review services, these are largely non-interoperable. The result of this, especially for post-publication services, is that most evaluations are difficult to discover, lost, or rarely available in an appropriate context or platform for re-use. To date, it seems that little effort has been focused on aggregating the content of these services, which hinders its recognition as a valuable community process and for additional evaluation or assessment decisions.\n\nWhile several new overlay journals are currently thriving, the history of their success is invariably limited, and most journals that experimented with the model returned to their traditional coupled roots (Priem & Hemminger, 2012). Axios Review was closed down in early 2017 due to a lack of uptake from researchers, with the founder stating: “I blame the lack of uptake on a deep inertia in the researcher community in adopting new workflows” (Davis, 2017). Finally, it is probably worth mentioning that not a single overlay journal appears to have emerged outside of physics and math (Priem & Hemminger, 2012). This is despite the fast growth of arXiv spin-offs like biorXiv, and potential layered peer review through services such as ScienceOpen or the recently launched Peer Community In (peercommunityin.org).\n\nCoupled with the demise of services such as Axios Review, the generally low uptake of decoupled peer review processes suggests the overall reluctance of many research communities to adapt outside of the traditional coupled model. In this section, we have discussed a range of different arguments, variably successful platforms, and surveys and reports about peer review. Taken together, these reveal an incredible amount of friction to experimenting with peer review beyond that which is typically and incorrectly viewed as the only way of doing it. This reluctance is emphasized in recent surveys, for instance the one by Ross-Hellauer (2017) suggests that while attitudes towards the principles of OPR are rapidly becoming more positive, faith in its execution is not. We can perhaps expect this divergence due to the rapid pace of innovation, which has not led to rigorous or longitudinal evidence that these models are superior to the traditional process at either a population or system-wide level. Cultural or social inertia, then, is defined by this cycle between low uptake and limited incentives and evidence. Perhaps more important is the general under-appreciation of this intimate relationship between social and technological barriers, that is undoubtedly required to overcome this cycle. The proliferation of social media over the last decade provides excellent examples of how digital communities can leverage new technologies for great effect.\n\n\n3 Potential future models\n\nAs we have discussed in detail above, there has been considerable technological innovation in peer review in the last decade, which is leading to critical examination of it as a social process. Much of this has been driven by the advent of Web 2.0 technologies and new social media platforms, and an overall shift towards a more open system of scholarly communication. Previous work in this arena has described features of a Reddit-like model, combined with additional personalized features of other social platforms, like Stack Exchange, Netflix, and Amazon (Yarkoni, 2012). Here, we develop upon this by considering additional traits of models such as Wikipedia, GitHub, and Blockchain, and discuss these in the rapidly evolving socio-technological environment for the present system of peer review. In any vision of the future of scholarly publishing (Kriegeskorte et al., 2012), the evolution of peer review and evaluation systems must be considered. Any future peer review platform or system would greatly benefit from considering the following key features:\n\n1. Quality control and moderation, possibly through openness and transparency;\n\n2. Certification via personalized reputation or performance metrics;\n\n3. Incentive structures to motivate and encourage engagement.\n\nWhile discussing a number of principles that should guide the implementation of novel platforms for evaluating scientific work, Yarkoni (2012) argued that many of the problems researchers face have already been successfully addressed by a range of non-research focused social Web applications. Therefore, developing next-generation platforms for scientific evaluations should focus on adapting the best currently used approaches for these rather than on innovating entirely new ones (Neylon & Wu, 2009; Priem & Hemminger, 2010; Yarkoni, 2012). One important element that will determine the success or failure of any such peer-to-peer reputation or evaluation system is a critical mass of researcher uptake. This has to be carefully balanced with the demands and uptakes of restricted scholarly communities, which have inherently different motivations and practices in peer review. A remaining issue is the aforementioned cultural inertia, which can lead to low adoption of anything innovative or disruptive to traditional workflows in research. This is a perfectly natural trait for communities, where ideas out-pace technological innovation, which in turn out-paces the development of social norms. Hence, rather than proposing an entirely new platform or model of peer review, our approach here is to consider the advantages and disadvantages of existing models and innovations in social services and technologies (Table 4). We then explore ways in which such traits can be adapted, combined, and applied to build a more effective and efficient peer review system, while potentially reducing friction to its uptake.\n\nNote that some of these are already employed, alone or in combination, by different research platforms.\n\nReddit (reddit.com) is an open-source, community-based platform where users submit comments and original or linked content, organized into thematic lists of subreddits. As Yarkoni (2012) noted, a thematic list of subreddits can be automatically generated for any peer review platform using keyword metadata generated from sources like the National Library of Medicine’s Medical Subject Heading (MeSH) ontology. Members, or redditors, can upvote or downvote any submissions based on quality and relevance, and publicly comment on all shared content. Individuals can subscribe to contribution lists, and articles can be organized by time (newest to oldest) or level of engagement. Quality control is invoked by moderation through subreddit mods, who can filter and remove inappropriate comments and links. A score is given for each link and comment as the sum of upvotes minus downvotes, thus providing an overall ranking system. At Reddit, highly scoring submissions are relatively ephemeral, with an automatic down-voting algorithm implemented that shifts them further down lists as new content is added, typically within 24 hours of initial posting.\n\n3.1.1 Reddit as an existing “journal” of science. The subreddit for Science (reddit.com/r/science) is a highly-moderated discussion channel, curated by at least 600 professional researchers and with more than 15 million subscribers at the time of writing. The forum has even been described as “The world’s largest 2-way dialogue between scientists and the public” (Owens, 2014). Contributors here can add flair to their posts as a way of thematically organizing them based on research discipline, analogous to the container function of a typical journal. Individuals can also have flair as a form of subject-specific credibility (i.e., a peer status) upon provision of proof of education in their topic. Public contributions from peers are subsequently stamped with a status and area of expertise, such as “Grad student\|Earth Sciences.”\n\nScientists already further engage with Reddit through science AMAs (Ask Me Anythings), which tend to be quite popular. However, the level of discourse provided in this is generally not equivalent in depth compared to that perceived for peer review, and is more akin to a form of science communication or public engagement with research. In this way, Reddit has the potential to drive enormous amounts of traffic to primary research and there even is a phenomenon known as the “Reddit hug of death”, whereby servers become overloaded and crash due to Reddit-based traffic. The /r/science subreddit is viewed as a venue for “scientists and lay audiences to openly discuss scientific ideas in a civilized and educational manner”, according to the organizer, Dr. Nathan Allen (Lee, 2015). As such, an additional appeal of this model is that it could increase the public level of scientific literacy and understanding.\n\n3.1.2 Reddit-style peer evaluation. The essential part of any Reddit-style model with potential parallels to peer review is that links to scientific research can be shared and ranked (upvoted or downvoted) by the community. All links or texts can be publicly discussed in terms of methods, context, and implications, similar to any post-publication commenting system. Such a process for peer review could essentially operate as an additional layer on top of a pre-print archive or repository, much like a social version of an overlay journal. Ultimately, a public commenting system like this could achieve the same depth of peer evaluation as the formal process, but as a crowd-sourced process. However, it is important to note here that this is a mode of instantaneous publication prior to peer review, with filtering through interaction occurring post-publication. Furthermore, comments can receive similar treatment to submitted content, in that they can be upvoted, downvoted, and further commented upon in a cascading process. An advantage of this is that multiple comment threads can form on single posts and viewers can track individual discussions. Here, the highest-ranked comments could simply be presented at the top of the thread, while those of lowest ranking remain at the bottom.\n\nIn theory, a subreddit could be created for any sub-topic within research, and a simple nested hierarchical taxonomy could make this as precise or broad as warranted by individual communities. Reddit allows any user to create their own subreddit, pending certain status achievements through platform engagement. In addition, this could be moderated externally through ORCID, similar to the approach taken by ScienceOpen, in which five items in a peer’s ORCID profile are required to perform a peer review; or in this case, create a new subreddit. Connection to a social network within academia, such as ORCID, further allows community validation, verification, and judgement of importance. For example, being able to see whether senior figures in a given field have read or upvoted certain threads can be highly influential in decisions to engage with that thread, and vice versa. A very similar process already occurs at the Self Journal of Science, where contributors have a choice of voting either “This article has reached scientific standards” or “This article still needs revisions”, with public disclosure of who has voted in either direction. Threaded commenting could also be implemented, as it is vital to the success of any collaborative filtering platform, and also provides a highly efficient corrective mechanism. Peer evaluation in this form emphasizes progress and research as a discourse over piecemeal publications or objects as part of a lengthier process. Such a system could be applied to other forms of scientific work, which includes code, data and images, thereby allowing contributors to claim credit for their full range of research outputs. Comments could be signed by default, pseudonymous, or anonymized until a contributor chooses to reveal their identity. If required, anonymized comments could be filtered out automatically by users. A key to this could be peer identity verification, which can be done at the back-end via email or integrated via ORCID.\n\n3.1.3 Translating engagement into prestige. Reddit karma points are awarded for sharing links and comments, and having these upvoted or downvoted by other registered members. The simplest implementation of such a voting system for peer review would be through interaction with any article in the database with a single click. This form of field-specific social recommendation for content simultaneously creates both a filter and a structured feed, similar to Facebook and Google+, and can easily be automated. With this, contributions get a rating, which accumulate to form a peer-based rating as a form of reputation and could be translates into a quantified level of community-granted prestige. Ratings are transparent and contributions and their ratings can be viewed on a public profile page. More sophisticated approaches could include graded ratings—e.g., five-point responses, like those used by Amazon—or separate rating dimensions providing peers with an immediate snapshot of the strengths and weaknesses of each article. Such a system is already in place at ScienceOpen, where referees evaluate an article for importance, validity, completeness, and comprehensibility using a five-star system. For any given set of articles retrieved from the database, a ranking algorithm could be used to dynamically order articles on the basis of a combination of quality (an article’s aggregate rating within the system, like at Stack Exchange), relevance (using a recommendation system akin to Amazon or ScienceOpen), and recency (newly added articles could receive a boost). By default, the same algorithm would be implemented for all peers, as on Reddit. The issue here is making any such karma points equivalent to the amount of effort required to obtain them, and also ensuring that they are valued by the broader research community and assessment bodies. This could be facilitated through a simple badge incentive system, such as that designed by the Center for Open Science for core open practices (cos.io/our-services/open-science-badges/).\n\n3.1.4 Can the wisdom of crowds work with peer review? One might consider a Reddit-style model as pitching quantity versus quality. Typically, comments provided on Reddit are not at the same level in terms of depth and rigor as those that we would expect from traditional peer review—as in, there is more to research evaluation than simply upvoting or downvoting. Furthermore, the range of expertise is highly variable due to the inclusion of specialists and non-specialists as equals (“peers”) within a single thread. However, there is no reason why a user prestige system akin to Reddit flair cannot be utilised to differentiate varying levels of expertise. The primary advantage here is that the number of participants is uncapped, therefore emphasizing the potential that Reddit has in scaling up participation in peer review. With a Reddit model, we must hold faith that sheer numbers will be sufficient in providing an optimal assessment of any given contribution and that any such assessment will ultimately provide a consensus of high quality and reusable results. Social review of this sort must therefore consider at what point is the process of review constrained in order to produce such a consensus, and one that is not self-selective as a factor of engagement rather than accuracy. This is termed the “Principle of Multiple Magnifications” by Kelty et al. (2008), which surmises that in spite of self-selectivity, more reviewers and more data about them will always be better than fewer reviewers and less data. The additional challenge, then, will be to capture and archive consensus points for external re-use. Journals such as F1000 Research already have such a tagging system, where reviewers can mark a submission as approved after peer review iterations.\n\n“The rich get richer” is one potential phenomenon for this style of system. Content from more prominent researchers may receive relatively more comments and ratings, and ultimately hype, as with any hierarchical system, including that for traditional scholarly publishing. Research from unknown authors may go relatively under-noticed and under-used, but will at least have been publicized. One solution to this is having a core community of editors, drawing on the r/science subreddit’s community of moderators. The editors could be empowered to invite peers to contribute to discussion threads, essentially wielding the same executive power as a journal editor, but combined with that of a forum moderator. Recent evidence suggests that such intelligent crowd reviewing has the potential to be an efficient and high quality process (List, 2017).\n\nAmazon was one of the first websites allowing the posting of public customer book reviews. The process is completely open and informal, so that anyone can write a review and vote, providing usually that they have purchased the product. Customer reviews of this sort are peer-generated product evaluations hosted on a third-party website, such as Amazon (Mudambi & Schuff, 2010). Here, usernames can be either real identities or pseudonyms. Reviews can also include images, and have a header summary. In addition, a fully searchable question and answer section on individual product pages allows users to ask specific questions, answered by the page creator, and voted on by the community. Top-voted answers are then displayed at the top. Chevalier & Mayzlin (2006) investigated the Amazon review system finding that, while reviews on the site tended to be more positive, negative reviews had a greater impact in determining sales. Reviews of this sort can therefore be thought of in terms of value addition or subtraction to a product or content, and ultimately can be used to guide a third-party evaluation of a product and purchase decision (i.e., a selectivity process).\n\n3.2.1 Amazon’s star-rating system. Star-rating systems are used frequently at a high-level in academia, and are commonly used to define research excellence, albeit perhaps in a flawed and an arguably detrimental way; e.g., the Research Excellence Framework in the UK (ref.ac.uk) (Mhurchú et al., 2017; Moore et al., 2017; Murphy & Sage, 2014). A study about Web 2.0 services and their use in alternative forms of scholarly communication by UK researchers found that nearly half (47%) of those surveyed expected that peer review would be complemented by citation and usage metrics and user ratings in the future (Procter et al., 2010a; Procter et al., 2010b). Amazon provides a sophisticated collaborative filtering system based on five-star ratings, usually combined with several lines of comments and timestamps. This system is summarized with the proportion of total customer reviews that have rated a product at each star level. An average star rating is also given for each piece of content. A low rating (one star) indicates an extremely negative view, whereas a high rating (five stars) reflects a positive view of the product. An intermediate scoring (three stars) can either represent a mid-view of a balance between negative and positive points, or merely reflect a nonchalant attitude towards a product. These ratings reveal fundamental details of accountability and are a sign of popularity and quality for items and sellers.\n\nThe utility of such a star-rating system for research is not immediately clear, or whether positive, moderate, or negative ratings would be more useful. A rating by itself would be a fairly useless design for researchers without being able to see the context and justification behind it. It is also unclear how a combined rate and review system would work for non-traditional research outputs, as the extremity and depth of reviews have been shown to vary depending on the type of content (Mudambi & Schuff, 2010). Furthermore, the ubiquitous five-star rating tool used across the Web is flawed in practice and produces highly skewed results. For one, when people rank products or write reviews online, they are more likely to leave positive feedback. The vast majority of ratings on YouTube, for instance, is five stars and it turns out that this is repeated across the Web with an overall average estimated at about 4.3 stars, no matter the object being rated (Crotty, 2009). Ware (2011) confirmed this average for articles rated in PLOS, suggesting that academic ranking systems operate in a similar manner to other social platforms. Ratings systems also select for popularity rather than quality, which is the opposite of what scholarly evaluation seeks (Ware, 2011). Another problem with commenting and rating systems is that they are open to gaming and manipulation. The Amazon system has been widely abused and it has been demonstrated how easy it is for an individual or small groups of friends to influence the popularity metrics even on hugely-visited websites like Time 100 (Emilsson, 2015; Harmon & Metaxas, 2010). Amazon has historically prohibited compensation for reviews, prosecuting businesses who pay for fake reviews as well as the individuals who write them. Yet, with the exception that reviewers could post an honest review in exchange for a free or discounted product as long as they disclosed that fact. A recent study of over seven million reviews indicated that the average rating for products with these incentivized reviews was higher than non-incentivized ones (Review Meta, 2016). Aiming to contain this phenomenon, Amazon has recently decided to adapt its Community Guidelines to eliminate incentivized reviews. As mentioned above, ScienceOpen offers a five-star rating system for papers, combined with post-publication peer review, but here the incentive is simply that the review content can be re-used, credited, and cited. How this translates to user and community perception in an academic environment remains an interesting question for further research.\n\n3.2.2 Reviewing the reviewers. At Amazon, users can vote whether or not a review was helpful with simple binary yes or no options. Potential abuse can also be reported and avoided here by creating a system of community-governed moderation. After a sufficient number of yes votes, a user is upgraded to a spotlight reviewer through what essentially is a popularity contest. As a result, their reviews are given more prominence. Top reviews are those which receive the most helpful upvotes, usually because they provide more detailed information about a product.\n\nOne potential way of improving rating and commenting systems is to weight such ratings according to the reputation of the rater (as done on Amazon, eBay, and Wikipedia). Reputation systems intend to achieve three things: foster good behavior, penalize bad behavior, and reduce the risk of harm to others as a result of bad behavior (Ubois, 2003). Key features are that reputation can rise and fall and that reputation is based on behavior rather than social connections, thus prioritizing engagement over popularity. In addition, reputation systems do not have to use the true names of the participants but, to be effective and robust, they must be tied to an enduring identity infrastructure. Frishauf (2009) proposed a reputation system for peer review in which the review would be undertaken by people of known reputation, thereby setting a quality threshold that could be integrated into any social review platform and automated (e.g., via ORCID). One further problem with reputation systems is that having a single formula to derive reputation leaves the system open to gaming, as with almost any process that can be measured and quantified. Gashler (2008) proposed a decentralized and secured system where each reviewer would digitally sign each paper, hence the digital signature would link the review with the paper. Such a web of reviewers and papers could be data mined to reveal information on the influence and connectedness of individual researchers within the research community. Depending on how the data were mined, this could be used as a reputation system or web-of-trust system that would be resistant to gaming because it would specify no particular metric.\n\nStack Exchange (stackexchange.com) is a collective intelligence system comprising multiple individual question and answer sites, many of which are already geared towards particular research communities, including maths and physics. The most popular site within Stack Exchange is Stack Overflow, a community of software developers and a place where professionals exchange problems, ideas, and solutions. Stack Exchange works by having users publish a specific problem, and then others contribute to a discussion on that issue. This format is considered to be a form of dynamic publishing by some (Heller et al., 2014). The appeal of Stack Exchange is that threaded discussions are often brief, concise, and geared towards solutions, all in a typical Web forum format. Highly regarded answers are positioned towards the top of threads, with others concatenated beneath. Like the Amazon model of weighted ratings, voting in Stack Exchange is more of a process that controls relative visibility. The result is a library of topical questions with high quality discussion threads and answers, developed by capturing the long tail of knowledge from communities of experts. The main distinction between this and scholarly publishing is that new material rarely is the focus of discussion threads. However, the ultimate goal remains the same: to improve knowledge and understanding of a particular issue. As such, Stack Exchange is about creating self-governing communities and a public, collaborative knowledge exchange forum based on software (Begel et al., 2013).\n\n3.3.1 Existing Overflow-style platforms. Some subject-specific platforms for research communities already exist that are similar to or based on Stack Exchange technology. These include BioStars (biostars.org), a rapidly growing Bioinformatics resource, the use of which has contributed to the completion of traditional peer reviewed publications (Parnell et al., 2011). Another is PhysicsOverflow, a platform for real-time discussions between physics professionals combined with an open peer review system (Pallavi Sudhir & Knöpfel, 2015). PhysicsOverflow forms the counterpart forum to MathOverflow (Tausczik et al., 2014), with both containing a graduate-level question and answer forum, and an Open Problems section for collaboration on research issues. Both have a Reviews section to complement formal journal-led peer review, where peers can submit preprints (e.g., from arXiv) for public peer evaluation, considered by most to be an “arXiv-2.0”. Responses are divided into reviews and comments, and given a score based on votes for originality and accuracy. Similar to Reddit, there are moderators but these are democratically elected by the community itself. Motivation for engaging with these platforms comes from a personal desire to assist colleagues, progress research, and receive recognition for it (Kubátová, 2012) – the same as that for peer review. Together, both have created open community-led collaboration and discussion platforms for their research disciplines.\n\n3.3.2 Community-granted reputation and prestige. One of the key features of Stack Exchange is that it has an inbuilt community-based reputation system, karma, similar to that for Reddit. Identified peers rate or endorse the contributions of others and can indicate whether those contributions are positive (useful or informative) or negative. This provides a point-based reputation system for individuals, based not just on the quantity of engagement with the platform and its peers alone, but also on the quality and relevance of those engagements, as assessed by the wider engaging community (stackoverflow.com/help/whats-reputation). Peers have their status and moderation privileges within the platform upgraded as they gain reputation. Such automated privilege administration provides a strong social incentive for engaging within the community. Furthermore, peers who asked the original questions mark answers considered to be the most correct, thereby acknowledging the most significant contributions while providing a stamp of trustworthiness. This has the additional consequence of reducing the strain of evaluation and information overload for other peers by facilitating more rapid decision making, a behavior based on simple cognitive heuristics (e.g., social influences such as the “bandwagon effect” and position bias) (Burghardt et al., 2017). Threads can also be closed once questions have been answered sufficiently, based on a community decision, which enables maximum gain of potential karma points. This terminates further contribution but ensures that the knowledge is captured for future needs.\n\nKarma and reputation can thus be achieved and incentivized by building and contributing to a growing community and providing knowledgeable and comprehensible answers on a specific topic. Within this system, reputation points are distributed based on social activities that are akin to peer review, such as answering questions, giving advice, providing feedback, providing data, and generally improving the quality of work in the open. The points directly reflect an individual’s contribution to that specific research community. Such processes ultimately have a very low barrier to entry, but also expose peer review to potential gamification through integration with a reputation engine, a social bias which proliferates through any technoculture (Belojevic et al., 2014).\n\n3.3.3 Badge acquisition on Stack Overflow. An additional important feature of Stack Overflow is the acquisition of merit badges, which provide public stamps of group affiliation, experience, authority, identity and goal setting (Halavais et al., 2014). These badges define a way of locally and qualitatively differentiating between peers, and also symbolize motivational learning targets to achieve (Rughiniş & Matei, 2013). Stack Overflow also has a system of tag badges to attribute subject-level expertise, awarded once a peer achieves a certain voting score. Together, these features open up a novel reputation system beyond traditional measurements based on publications and citations, that can also be used as an indication of expertise transferable beyond the platform itself. As such, a Stack Exchange model can increase the mobility of researchers who contribute in non-conventional ways (e.g., through software, code, teaching, data, art) and are based at non-academic institutes. There is substantial scope in creating a reputation platform that goes beyond traditional measurements to include social network influence and open peer-to-peer engagement. Ultimately, this model can potentially transform the diversity of contributors to professional research and level the playing field for all types of formal contribution.\n\nGit is an open-source distributed version control system developed by the Linux community in 2005. GitHub, launched in 2008, works as a Web-based Git service and has become the de facto social coding platform for collaborative and open source development and code sharing (Kosner, 2012; Thung et al., 2013). It holds many potentially desirable features that might be transferable to a system of peer review (von Muhlen, 2011), such as its openness, version control and project management functionality, and system of accreditation and attribution for contributions. Despite its capability for not just sharing code, but also executable papers that automatically knit together text, data, and analyses into a living document, the true power of GitHub appears to be acknowledged infrequently by academic researchers (Priem, 2013).\n\n3.4.1 Social functions of GitHub. Software review is an important part of software development, particularly for collaborative efforts. It is important that contributions are reviewed before they are merged into a code base, and GitHub provides this functionality. In addition, GitHub offers the ability to discuss specific issues, where multiple people can contribute to such a discussion, and discussions can refer to code segments or code changes and vice versa. GitHub also includes a variety of notification options for both users and project repositories. Users can watch repositories or files of interest and be notified of any new issues or commits (updates), and someone who has discussed an issue can also be notified of any new discussions of that same issue. Issues can also be tagged (labelled in a manner that allows grouping of multiple issues with the same tag), and assigned to one or more participants, who are then responsible for that issue. Another item that GitHub supports is a checklist, a set of items that have a binary state, which can be used to implement and store the status of a set of actions. GitHub also allows users to form organizations as a way of grouping contributors together to manage access to different repositories. All contributions are made public as a way for users to obtain merit.\n\nPrestige at GitHub can be further measured quantitatively as a social product through the star-rating system, which is derived from the number of followers or watchers and the number of times a repository has been forked (i.e., copied) or commented on. This could ultimately shift the power dynamic in deciding what gets viewed and re-used away from editors, journals, or publishers to individual researchers. This then actually leverages a new mode of prestige, which is conferred through how work is engaged with by the wider community and not by the packaging in which it is contained (analogous to the prestige associated with journal brands).\n\nGiven these properties, it is clear that GitHub could be used to implement some style of peer evaluation and that it is well-suited to fine-grained iteration between reviewers and authors (Ghosh et al., 2012), given that all parties are identified. Making peer review a social process by distributing reviews to numerous peers, divides the burden and allows individuals to focus on their particular area of expertise. Peer review would operate more like a social network, with specific tasks (or repositories) being developed, distributed, and promoted through GitHub. As all code and data are supplied, peers would be able to assess methods and results comprehensively, which increases rigor, transparency, and replicability. Reviewers would also be able to claim credit and be acknowledged for their tracked contributions, and thereby quantify their impact on a project as a supply of individual prestige. This in turn facilitates an assessment of quality of reviews and reviewers. As such, evaluation becomes an interactive and dynamic process, with version control facilitating this all in a post-publication environment (Ghosh et al., 2012). The potential issue of proliferating non-significant work here is minimal, as projects that are not deemed to be interesting or of a sufficient standard of quality are simply never paid attention to in terms of follows, contributions, and re-use.\n\n3.4.2 Current use of GitHub for peer review. An example use of GitHub for peer review already exists in The Journal of Open Source Software (JOSS, joss.theoj.org). JOSS provides a lightweight mechanism for software developers to quickly supplement their code with metadata and a descriptive paper, and then to submit this package for review and publication. ReScience (rescience.github.io) is another GitHub-based journal, created to publish replication efforts in computational science.\n\nHere is a summary of how JOSS uses GitHub: The JOSS submission webpage converts a submission into a new GitHub issue of type “pre-review” in the JOSS-review repository (github.com/openjournals/joss-reviews). The editor-in-chief checks a submission, and if deemed suitable for review, assigns it to a topic editor who in turn assigns it to one or more reviewers. The topic editor then issues a command that creates a new issue of type “review”, with a check-list of required elements for the review. Each reviewer performs their review by checking off elements of the review issue with which they are satisfied. When they feel the submitter needs to make changes to make an element of the submission acceptable, they can either add a new comment in the review issue, which the submitter will see immediately, or they can create a new issue in the repository where the submitted software and paper exist—which could also be on GitHub, but is not required to be—and reference said issue in the review. In either case, the submitter is automatically and immediately notified of the issue, prompting them to address the particular concern raised. This process can iterate repeatedly, as the goal of JOSS is not to reject submissions but to work with submitters until their submissions are deemed acceptable. If there is a dispute, the topic editor (as well as the main editor, other topic editors, and anyone else who chooses to follow the issue) can weigh in. At the end of this process, when all items in the review check-list are resolved, the submission is accepted by the editor and the review issue is closed. However, it is still available and is linked from the accepted (and now published) submission. A good future option for this style of model could be to develop host-neutral standards using Git for peer review. For example, this could be applied by simply using a prescribed directory structure, such as: manuscript_version_1/peer_reviews, with open commenting via the issues function.\n\nWikipedia is the freely available, multi-lingual, expandable encyclopedia of human knowledge. Wikipedia, like Stack Exchange, is another collective intelligence and authoring system whereby contributing communities are essentially unlimited in scope. Under a constant and instantaneous process of reworking and updating, new articles are added on a daily basis. Wikipedia operates through a system of collective intelligence based on linking knowledge workers through social media (Kubátová, 2012). Contributors to Wikipedia are largely anonymous volunteers, who are encouraged to participate mostly based on the principles guiding the platform (e.g., altruistic knowledge generation), and therefore often for reasons of personal satisfaction. Edits occur as cumulative and iterative improvements, and due to such a collaborative model, explicitly defining authorship becomes a complex task. Moderation and quality control is provided by a community of experienced editors and software-facilitated removal of mistakes, which can also help to resolve conflicts caused by concurrent editing by multiple authors (wikipedia.org/wiki/Help:Edit_conflict). Platforms already exist that enable multiple authors to collaborate on a single document in real time, including Overleaf and Authorea, which highlights the potential for this model to be extended into peer review. Communities of moderators at Wikipedia also functionally exercise editorial power over content, and are nominated using conventional elections that variably account for their standing reputation. The apparent “free for all” appearance of Wikipedia is actually more of a system of governance, based on implicitly shared values in the context of what is perceived to be useful for consumers, and transformed into operational rules to moderate the quality of content (Kelty et al., 2008).\n\n3.5.1 “Peers” and “reviews” in a wiki-world. Wikipedia already has its own mode of peer review, requested by anyone as a way to receive ideas on how to improve articles that are already considered to be “decent” (en.wikipedia.org/wiki/Wikipedia:Peer_review/guidelines). It can be used for nominating potentially good articles that could become candidates for a featured article. Featured articles are considered to be the best articles Wikipedia has to offer, as determined by its editors and the fact that only ∼0.1% are featured, or an article of any grade. Users submitting a new request are encouraged to review an article from those already listed, and encourage reviewers by replying promptly and appreciatively to comments. Compared to the conventional peer review process, where experts themselves participate in reviewing the work of another, the majority of the volunteers here, like most editors in Wikipedia, lack formal expertise in the subject at hand (Xiao & Askin, 2012). This is considered to be a positive thing within the Wikipedia community, as it can make technically-worded articles more accessible to non-specialist readers.\n\nThis process clearly lacks the “peer” aspect of peer review, which can potentially lead to propagation of factual errors (e.g., Hasty et al. (2014)). This creates a general perception of low quality from the research community, in spite of difficulties in actually measuring this (Hu et al., 2007). However, as was originally the case with Open Access publishing, much of this perception can most likely be explained by a lack of familiarity with the model, and we might expect comfort to increase and attitudes to change with increased engagement and understanding of the process (Xiao & Askin, 2014). If seeking expert input, users can invite editors from a subject-specific volunteers list or notify relevant WikiProjects. Furthermore, Wikipedia articles never “pass” a review, which, although part of the process of conventional validation, is of little actual value on the platform due to its dynamic nature. Indeed, wikicommunities appear to have distinct values to academic communities, being based more on inclusive community participation and mediation than on trust, exclusivity, and identification (Wang & Wei, 2011). Therefore, the process is perhaps best viewed as a process of “peer production”, but where attainment of the level of peer is relatively lower to that of an accredited expert. This provides a difference in community standing for Wikipedia content, with value being conveyed through contemporariness, mediation of debate, and transparency of information, rather than any perception of authority as with traditional scholarly works (Black, 2008). Such a process could be feasibly combined with trust metrics for verification, developed for sociology and psychology to describe the relative standing of groups or individuals in virtual communities (en.wikipedia.org/wiki/Trust_metric).\n\n3.5.2 Democratization of peer review. The advantage of Wikipedia over traditional review-then-publish processes comes from the fact that articles are enhanced consistently as new articles are integrated, statements are reworded, and factual errors are corrected as a form of iterative bootstrapping. Therefore, while one might consider a Wikipedia page to be of insufficient quality relative to a peer reviewed article at a given moment in time, this does not preclude it from meeting that quality threshold in the future. Therefore, Wikipedia might be viewed as an information trade-off between accuracy and scale, but with a gap that is consistently being closed as the overall quality improves. Another major statement that a Wikipedia-style of peer review makes is that rather than being exclusive, it is an inclusive process that anyone is allowed to participate in and the barriers to entry are very low—anyone can potentially be granted peer status and participate in the debate and vetting of knowledge. Wikipedia represents a fairly extreme alternative to peer review with a relatively large pool of potential, when traditionally the barriers to entry for peer review are very high, and overcoming these is based on expertise. (Kelty et al., 2008). This represents an enormous shift from the generally technocratic process of conventional peer review to one that is inherently more democratic. However, while the number of contributors is very large, more than 30 million, one third of all edits are made by only 10,000 people, just 0.03% (en.wikipedia.org/wiki/Wikipedia:List_of_Wikipedians_by_number_of_edits).\n\nThis is broadly similar to what is observed in current academic peer review systems, where the majority of the work is performed by a minority of the participants (Fox et al., 2017; Gropp et al., 2017; Kovanis et al., 2016). Any wiki-based peer review system would also alleviate the increasing burden on editors by distributing the endeavor more efficiently among members of the wider community—a high-risk, high-gain approach to generating academic capital (Black, 2008). A possible risk is the creation of a highly conservative network of norms due to the governance structure, which could end up being even more bureaucratic and create community silos rather than coherence (Heaberlin & DeDeo, 2016). To date, attempts at implementing a Wikipedia-like editing strategy for journals have been largely unsuccessful (e.g., at Nature (Zamiska, 2006)). There are intrinsic differences in authority models used in Wikipedia communities (where the validity of the end result derives from verifiability, not personal authority of authors and reviewers) that would need to be aligned with the norms and expectations of science communities. In the latter, author statements and peer reviews are considered valid because of the personal, identifiable status and reputation of authors, reviewers and editors, which could be feasibly combined with Wikipedia review models into a single solution. However, a more rigorous editorial review process is the reason why the original form of Wikipedia, known as Nupedia, ultimately failed (Sanger, 2005). Future developments of any Wikipedia-like peer review tool could expect strong resistance from universities due to potential disruption to assessment criteria, funding assignment, and intellectual property, as well as from commercial publishers since academics would be releasing their research to the public for free instead of to them.\n\nHypothesis is a lightweight, portable Web annotation tool that operates across publishing platforms (Perkel, 2015), ambitiously described as a “peer review layer for the entire Internet” (Farley, 2011). It relies on pre-existing published content to function, similar to other annotation services, such as PubPeer and PaperHive. Annotation is a process of enriching research objects through the addition of knowledge, and also provides an interactive educational opportunity by raising questions and creating opportunities to collect the perspectives of multiple peers in a single venue; providing a dual functionality for collaborative reading and writing. Web annotation services like Hypothesis allow annotations (such as comments or peer reviews) to live alongside the content but also separate from it, allowing communities to form and spread across the internet and across content types, such as HTML, PDF, EPUB, or other formats (Whaley, 2017). Further, as of February 2017, annotation became a Web standard recognized by the Web Annotation Working Group, W3C (2017). Under this model of Web annotation described by the W3C, annotations belong to and are controlled by the user rather than any individual publisher or content host. Users use a bookmarklet or browser extension to annotate any webpage they wish, and form a community of Web citizens.\n\nHypothesis permits the creation of public, group private, and individual private annotations, and is therefore compatible with a range of open and closed peer review models. Web annotation services not only extend peer review from academic and scholarly content to the whole Web, but open up the ability to annotate to any Web-browser. While the platform concentrates on focus groups within publishing, journalism, and academia, Hypothesis offers a new way to enrich, fact check, and collaborate on online content. Unlike Wikipedia, the core content never changes but the annotations are viewed as an overlay service on top of static content. This also means that annotations can be made at any time during the publishing process, including the pre-print stage. Reviewers often provide annotated versions of submitted manuscripts during conventional peer review, and Web annotation is part of the digitization of this process, while also decoupling it from journal hosts. A further benefit of Web annotations is that they are precise, since they can be applied in line rather than at the end of an article as is the case with formal commenting.\n\nAnnotations have the potential to enable new kinds of workflows where editors, authors, and reviewers all participate in conversations focussed on research manuscripts or other digital objects, either in a closed or public environment (Vitolo et al., 2015). At the present, activity performed by Hypothesis and other Web annotation services is poorly recognized in scholarly communities, although such activities can be tied to ORCID. However, there is definite value in services such as PubPeer, an online community mostly used for identifying cases of academic misconduct and fraud, perhaps best known for its user-led post-publication critique of a Nature paper on STAP (Stimulus-Triggered Acquisition of Pluripotency) cells. This ultimately prompted the formal retraction of the paper, demonstrating that post-publication annotation and peer review, as a form of self-correction and fraud detection, can out-perform that of the conventional pre-publication process. PubPeer has also been leveraged as a way to mass-report post-publication checks for the soundness of statistical analyses. One large-scale analysis using a tool called statcheck was used to post 50,000 annotations on the psychological literature (Singh Chawla, 2016), as a form of large-scale public audit for published research.\n\nPeer review has the potential to be reinvented as a more efficient, fair, and otherwise attribute-enabled process through blockchains, a computer data structure that operates a distributed public ledger. A blockchain connects a row of data blocks through a cryptographic function, with each block containing a time stamp and a link to the previous block in the chain. This system is decentralized, distributed, immutable, and transparent (Antonopoulos, 2014; Nakamoto, 2008; Yli-Huumo et al., 2016). Perhaps most importantly, individual chains are managed by peer-to-peer networks that collectively adhere to specific validation protocols. Blockchain became widely known as the data structure in Bitcoin due to its ability to efficiently record transactions between parties in a verifiable and permanent manner. It has also been applied to other uses including sharing verified business transactions, proof of ownership of legal documents, and distributed cloud storage.\n\nThe blockchain technology could be leveraged to create a tokenized peer review system involving penalties for members who do no uphold the adopted standards and vice versa. A blockchain-powered peer-reviewed journal could be issued as a token system to reward contributors, reviewers, editors, commentators, forum participants, advisors, staff, consultants, and indirect service providers involved in scientific publishing (Swan, 2015). Such rewards could be in the form of reputation and/or remuneration, potentially through a form of digital currency (say Science Coins). Through a system of community trust, blockchains could be used to handle the following tasks:\n\n1. Authenticating scientific papers (using time stamps and checksums), combating fraudulent science;\n\n2. Allowing and encouraging reviewers to actively engage in the scientific community;\n\n3. Rewarding reviewers for peer reviews with Science Coins;\n\n4. Allowing authors to contribute by giving Science Coins;\n\n5. Supporting verification and replicability of research.\n\n6. Keeping reviewers and authors anonymous, while providing a validated certification of their identity as researchers, and rewarding them.\n\nThis could help to improve the quality and responsiveness of peer reviews, as these are published publicly and the different participants are rewarded for their contributions. For instance, reviewers for a blockchain-powered peer-reviewed journal could invest tokens in their comments and get rewarded if the comment is upvoted by other reviewers and the authors. All tokens need to be spent in making comments or upvoting other comments. When the peer review is completed, reviewers get rewarded according to the quality of their remarks. In addition, the rewards can be attributed even if reviewer and author identity is kept secret; such a system can decouple the quality assessment of the reviews from the reviews themselves, such that reviewers get credited while their reviews are kept anonymous. Moreover, increased transparency and interaction is facilitated between authors, reviewers, the scientific community, and the public. The journal Ledger, launched in 2015, is the first academic journal that makes use of a system of digital signatures and time stamps based on blockchain technology (ledgerjournal.org). The aim is to generate irrevocable proof that a given manuscript existed on the date of publication.\n\nFurthermore, blockchain-based models offer the potential to go well beyond peer review, possibly integrating all functions of publication in general. They could be used to support data publication, research evaluation, incentivization, and research fund distribution. A relevant example is a proposed decentralized peer review group as a way of managing quality control in peer review via blockchain through a system of cohort-based training (Dhillon, 2016). This has also been leveraged as a “proof of existence” platform for scientific research (Torpey, 2015) and medical trials (Carlisle, 2014). However, the uptake from the academic community remains low thus far, despite claims that it could be a potential technical fix to the reproducibility crisis in research (Bartling & Fecher, 2016). As with other novel processes, this is likely due to broad-scale unfamiliarity with blockchain, and perhaps even discomfort due to its financial association with Bitcoin.\n\nAnother frontier is the advent of machine learning (ML) and neural network tools that may potentially assist with the peer review process. Machine learning, as a technique, is rapidly becoming a service that can be utilized at a low cost by an increasing number of individuals. For example, Amazon now provides ML as a service through their Amazon Web Services platform, Google released their ML framework, TensorFlow, and Facebook have similarly contributed code of their Torch scientific learning framework. ML has been very widely adopted in tackling various challenges, including image recognition, content recommendation, fraud detection, and energy optimization. In higher education, adoption has been limited to automated evaluation of teaching and assessment, and in particular for plagiarism detection. The primary benefits of Web-based peer assessment are limiting peer pressure, reducing management workload, increasing student collaboration and engagement, and improving the understanding of peers as to what critical assessment procedures involve (Li et al., 2009).\n\nThe same is approximately true for using computer-based automation for peer review, for which there are three main practical applications. The first is determining whether a piece of work under consideration meets the minimal requirements of the process to which it has been submitted (i.e., for recommendation). For example, does a clinical trial contain the appropriate registration information, are the appropriate consent statements in place, have new taxonomic names been registered, and does the research fit in with the existing body of published literature (Sobkowicz, 2008)? The computer might also look at consistency through the paper (for example searching for statistical error or method description incompleteness: if there is a multiple group comparison, is the p-value correction algorithm indicated?) This might be performed using a simpler text mining approach, as is performed by statcheck (Singh Chawla, 2016). Under normal technical review, these criteria need to be (or should be) checked manually either at the editorial submission stage or at the review stage. ML techniques can automatically scan documents to determine if the required elements are in place, and can generate an automated report to assist review and editorial panels, facilitating the work of the human reviewers. Moreover, any relevant papers can be automatically added to the editorial request to review, enabling referees to automatically have a greater awareness of the wider context of the research. This could also aid in preprint publication before manual peer review occurs.\n\nThe second approach is to automatically determine the most appropriate reviewers for a submitted manuscript, by using a co-authorship network data structure (Rodriguez & Bollen, 2008). The advantage of this is that it opens up the potential pool of referees beyond whom is simply known by an editor or editorial board. Removing human-intervention from this part of the process reduces potential biases (e.g., author recommended exclusion or preference) and can automatically identify potential conflicts of interest (Khan, 2012). Dall’Aglio (2006) suggests ways this algorithm could be improved, for example through cognitive filtering to automatically analyze text and compare that to editor profiles as the basis for assignment. This could be built upon for referee selection by using an algorithm based on social networks, which can also be weighted according to the influence and quality of participant evaluations (Rodriguez et al., 2006), and referees can be further weighted based on their previous experience and contributions to peer review and their relevant expertise, thereby providing a way to train and develop the identification algorithm.\n\nFinally, given that machine-driven research has been used to generate substantial and significant novel results based on ML and neural networks, we should not be surprised if, in the future, they can have some form of predictive utility in the identification of novel results during peer review. In such a case, machine learning would be used to predict the future impact of a given work (e.g., future citation counts), and in effect to do the job of impact analysis and decision making instead of or alongside a human reviewer. We have to keep a close watch on this potential shift in practice as it comes with obvious potential pitfall by encouraging even more editorial selectivity, especially when network analysis is involved. For example, research in which a low citation future is predicted would be more susceptible to rejection, irrespective of the inherent value of that research. Conversely, submissions with a high predicted citation impact would be given preferential treatment by editors and reviewers. Caution in any pre-publication judgements of research should therefore always be adopted, and not be used as a surrogate for assessing the real world impact of research through time. Machine learning is not about providing a total replacement for human input to peer review, but more how different tasks could be delegated or refined through automation.\n\nSome platforms already incorporate such methods for a variety of purposes. Scholastica (scholasticahq.com) includes real-time journal performance analytics that can be used to assess and improve the peer review process. Elsevier uses a system called Evise (elsevier.com/editors/evise) to check for plagiarism, recommend reviewers, and verify author profile information by linking to Scopus. The Journal of High Energy Physics uses automatic assignment to editors based on a keyword-driven algorithm (Dall’Aglio, 2006). This process has the potential to be entirely independent from journals and can be easily implemented as an overlay function for repositories, including pre-print servers. As such, it can be leveraged for a decoupled peer review process by combining certification with distribution and communication. It is entirely feasible for this to be implemented on a system-wide scale, with researcher databases such as ORCID becoming increasingly widely adopted. However, as the scale of such an initiative increases, the risk of over-fitting also increases due to the inherent complexity in modelling the diversity of research communities, although there are established techniques to avoid this. Questions have been raised about the impact of such systems on the practice of scholarly writing, such as how authors may change their approach when they know their manuscript is being evaluated by a machine (Hukkinen, 2017), or how machine assessment could discover unfounded authority in statements by authors through analysis of citation networks (Greenberg, 2009). One additional potential drawback of automation of this sort is the possibility for detection of false positives that might discourage authors from submitting.\n\nFinally, it is important to note that ML and neural networks are largely considered to be conformist, so they have to be used with care (Szegedy et al., 2014), and perhaps only for recommendations rather than decision making. The question is not about whether automation produces error, but whether it produces less error than a system solely governed by human interaction. And if it does, how does this factor in relation to the benefits of efficiency and potential overhead cost reduction? Nevertheless, automation can potentially resolve many of the technical issues associated with peer review and there is great scope for increasing the breadth of automation in the future. Initiatives such as Meta, an AI tool that searches scientific papers to predict the trajectory of research (meta.com), highlight the great promise of artificial intelligence in research and for application to peer review.\n\nPeer review has also evolved when used outside of traditional text-based scholarly publications to a wider variety of research outputs, policies, processes, and even people. These non-text products are increasingly being recognized as intellectual contributions to the research ecosystem. In order for the creators (authors) of these products to receive academic credit, they must currently be integrated into the publication system that forms the basis for academic assessment and evaluation. Peer review of methodologies, such as protocols.io (protocols.io), allows for detailed OPR of methods while also promoting reproducibility and refinement of techniques. This can help other scholars to begin work on related projects and test methodologies due to the openness of both the protocols themselves and the comments on them (Teytelman et al., 2016). Digital humanities projects, which include visualizations, text processing, mapping, and many other varied outputs, have been a subject for re-evaluating the role of peer review, especially for the purpose of tenure and evaluation (Ball et al., 2016). In 2006, the Modern Languages Association released a statement on the peer review and evaluation of new forms of scholarship, insisting that they “be assessed with the same rigor used to judge scholarly quality in print media” (Stanton et al., 2007). Fitzpatrick (2011a) considered the idea of an objective evaluation of non-text products in the humanities, as well as the challenges faced during evaluation of a digital product that may have much more to review than a traditional text product, including community engagement and sustainability practices. To work with these non-text products, humanities scholars have used multiple methods of peer review and embraced OPR in order to adapt to the increased creation of non-text, multimedia scholarly products, and to integrate these products into the scholarly record and review process (Anderson & McPherson, 2011).\n\n3.9.1 Software peer review. Software represents another area where traditional peer review has evolved. In software, peer review of code has been a standard part in computationally-intensive research for many years, particularly as a post-software creation check. Additionally, peer-programming (also known as pair-programming) has been growing in popularity, especially as part of the Agile methodology, where it is employed as a check made during software creation (Lui & Chan, 2006). Software development and sharing platforms, such as GitHub, support and encourage social code review, which can be viewed as a form of peer review that takes place both during creation and afterwards. However, developed software has not traditionally been considered an academic product for the purpose of hiring, tenure, and promotion. Likewise, this form of evaluation has not been formally recognized as peer review by the academic community yet.\n\nWhen it comes to software development, there is a dichotomy of review practices. On one hand, software developed in open source communities (not all software is released as open source; some is kept as proprietary for commercial reasons) relies on peer review as an intrinsic part of its existence, from creation and through continual evolution. On the other hand, software created in academia is typically not subjected to the same level of scrutiny. At present, there is no requirement for software, used to produce academic publications, to be released as part of the publication process, let alone be closely checked as part of the review process, though this may be changing due to government mandates and community concerns about reproducibility. One example from Computer Science is ACM SIGPLAN’s Programming Language Design and Implementation conference that encourages the submission of supporting material (including code) for review by a separate technical committee. Papers with successfully evaluated artifacts get stamped with seals of approval visible in the conference proceedings. ACM is adopting a similar strategy on a wider scale through its Task Force on Data, Software, and Reproducibility in Publication (acm.org/data-software-reproducibility).\n\nAcademic code is sometimes released as open source, and many such released codebases have led to remarkable positive changes, with prominent examples including the Berkeley Software Distribution (BSD), upon which the Mac operating system (MacOS) is built; the ubiquitous TCP/IP Internet protocol stack; the Squid web proxy; the Xen hypervisor, which underpins many cloud computing infrastructures; Spark, the big data stream processing framework; and the Weka machine learning suite.\n\nIn order to gain recognition for their software work, authors initially made as few changes to the existing system as possible and simply wrote traditional papers about their software, which became acceptable in an increasing number of journals over time (see the extensive list compiled by the UK’s Software Sustainability Institute: software.ac.uk/whichjournals-should-i-publish-my-software). At first, peer review for these software articles was the same as for any other paper, but this is changing now, particularly as journals specializing in software (e.g., SoftwareX (journals.elsevier.com/softwarex), the Journal of Open Research Software (JORS, openresearchsoftware.metajnl.com), the Journal of Open Source Software (JOSS, joss.theoj.org)) are emerging. The material that is reviewed for these journals is both the text and the software. For SoftwareX (elsevier.com/authors/authorservices/research-elements/software-articles/original-software-publications#submission) and JORS (openresearchsoftware.metajnl.com/about/#q4), the text and the software are reviewed equally. For JOSS, the review process is more focused on the software (based on the rOpenSci model (Ross et al., 2016) and less on the text, which is intended to be minimal (joss.theoj.org/about#reviewer_guidelines).\n\nThe purpose of the review also varies across these journals. In SoftwareX and JORS, the goal of the review is to decide if the paper is acceptable and to improve it through a non-public editor-mediated iteration with the authors and the anonymous reviewers. While in JOSS, the goal is to accept most papers after improving them if needed, with the reviewers and authors ideally communicating directly and publicly through GitHub issues. Although submitting source code is still not required for most peer review processes, attitudes are slowly changing. As such, authors increasingly publish works presented at major conferences (which are the main channel of dissemination in computer science) as open source.\n\n3.9.2 Data peer review. Many journals in biological science already ask authors to release data to reviewers when they submit a paper (e.g., Journal of Cell Science, Microbial Genomics, Royal Society Open Science), and journals can ask reviewers to say whether or not they have reviewed the data themselves. Making data available to reviewers and editors can help to correct obvious errors and, more importantly, serves as a strong incentive for authors to check data and analyses thoroughly before releasing them. For example, errors in datasets, improper data archiving standards, or simply a reluctance to release datasets can play a role in whether a particular journal rejects a paper. If journals enforced this more strictly, an improvement in data archiving standards would likely be the consequence. However, some reviewers, who may already be overburdened, might feel that checking data accuracy while reviewing the manuscript at the same time and receiving only little to no reward for their work (see Section 2.2) is unfair. The Peer Reviewers’ Openness Initiative (opennessinitiative.org/the-initiative) states that all data should be made publicly available for the purposes of evaluation and reproduction, and indicates that there is wider scope for the development of data peer review in the future (Morey et al., 2016).\n\nWhile individual publishers may use specific peer review methods when peer review is controlled by the author of the document to be reviewed, multiple peer review models can be used either in series or in parallel. For example, the FORCE11 Software Citation Working Group used a series of three different peer review models and methods to iteratively improve their principles document, leading to a journal publication (Smith et al., 2016). Initially, the document that was produced was made public and reviewed by GitHub issues (github.com/force11/force11-scwg [see Section 3.4]). The next version of the document was placed on a website, and new reviewers commented on it both through additional GitHub issues and through Hypothesis (via.hypothes.is/https://www.force11.org/software-citation-principles [see Section 3.6]). Finally, the document was submitted to PeerJ Computer Science, which used a pre-publication review process that allowed reviewers to sign their reviews and the reviews to be made public along with the paper authors’ responses after the final paper was accepted and published (Klyne, 2016; Kuhn, 2016a; Kuhn, 2016b). The authors also included an appendix that summarized the reviews and responses from the second phase. In summary, this document underwent three sequential and non-conflicting review processes and methods, where the second one was actually a parallel combination of two mechanisms.\n\nUsing such hybrid evaluation methods can prove to be quite successful, not just for reforming the peer review process but also to improve the impact of scientific publications. One could envision such a hybrid system with elements from the different models we discussed. Stack Overflow helps to surface practical solutions and their trade-offs. As such, it is well geared for research in practice, collating the community’s knowledge through collective validation, comparison, and innovation. Reddit-style discussion threads, which are characterized as being general and loose (not focused), are very effective for public engagement and for hosting virtual conferences or parallel discussion forums, and augmenting “physical” conferences. It might be necessary to adjust the Reddit karma system to give more weight to comments rather than new posts, to reduce the incentive to publish frequent, yet potentially low quality work.\n\n\n4 A hybrid peer review platform\n\nIn Section 3, we summarized the positive and negative traits of a range of individual existing social platforms. Each of these traits can be applied to address specific social or technical criticisms of conventional peer review, as outlined in Section 2. Many of them are overlapping and can be modeled into, and leveraged for, a single hybrid platform. The advantage is that they each relate to the core non-independent features required for any modern peer review process or platform: quality control, certification, and incentivization. Only by harmonizing all three of these, while grounding development in diverse community stakeholder engagement, can the implementation of any future model of peer review be ultimately successful. Such a system has the potential to greatly disrupt the current coupling between peer review and journals, and lead to an overhaul of scholarly communication to become one that is fit for the modern scholarly research environment.\n\nQuality control is the core function of peer review. Typically, this has been administered in a closed system, where editorial management formed the basis. A strong coupling of peer review to journals plays an important part in this, due to the association of researcher prestige with journal brand. By looking at platforms such as Wikipedia and Reddit, it is clear that community self-organization and governance represent a possible alternative when combined with a core community of moderators. These moderators would have the same operational functionality as editors in terms of gate-keeping and facilitating the process of engagement, but combined with the role of a Web forum moderator. Research communities could elect groups of moderators based on expertise, prior engagement with peer review, and transparent assessment of their reputation. This layer of moderation could be fully transparent in terms of identity by using persistent identifiers such as ORCID. Different communities could have different norms and procedures to govern content and engagement, and to self-organize into individual but connected platforms, similar to Stack Exchange or Reddit. ORCID has a further potential role of providing the possibility for a public archive of researcher information and metadata (e.g., publishing histories) that can be leveraged using automated techniques to match potential referees to items of interest, while avoiding conflicts of interest.\n\nIn such a system, published objects could be pre-prints, data, code, or any other digital research output. If these are combined with management through version control, similar to GitHub, quality control is provided by having a system of automated but managed invited review, public interaction and collaboration (like with Stack Exchange), and transparent refinement. This would also help prevent a situation where “the rich get richer”, as semi-automation ensures that all content has the same chance of being interacted with. Engagement could be conducted via a system of issues and public comments, as on GitHub, where the process is not to reject submissions, but to provide a system of constant improvement. Such a system is already implemented successfully at JOSS. Both community moderation and crowd sourcing would play an important role here to prevent underdeveloped feedback that is not constructive and could delay efficient manuscript progress. This could be further integrated with a blockchain process so that each addition to the process is transparent and verifiable.\n\nWhen authors and moderators deem the review process to have been sufficient for an object to have reached a community-decided level of quality or acceptance, threads can be closed (but remain public with the possibility of being re-opened, similar to GitHub issues), indexed, and the latest version is assigned a persistent identifier, such as a CrossRef DOI, as well as an appropriate license. If desired, these objects could then form the basis for submissions to journals, perhaps even fast-tracking them as the communication and quality control would already have been completed. Such a process would promote inclusive participation, community interaction, and quality would become a function of how information is engaged with, digested, and reused. The role of peer review would then be coupled with the concept of a “living published unit”, independent of journals themselves. The role of journals and publishers would be dependent on how well they justify their added value, once communitywide and public dissemination and peer review have been decoupled from them.\n\nThe current peer review process is generally poorly recognized as a scholarly activity. It remains quite imbalanced between publishers who receive financial gain for organising it and researchers who receive little or no compensation for performing it. Opacity in the peer review process provides a way for others to capitalize on it, as this provides a mechanism for those managing it, rather than performing it, to take credit in one form or another. This explains at least in part why there is resistance from many publishers in providing any form of substantive recognition to peer reviewers. Exposing the process, decoupling it from journals and providing appropriate recognition to those involved helps to return peer review to its synergistic, intra-community origin. Performance metrics provide a way of certifying the peer review process, and provide the basis for incentivizing engagement. As outlined above, a fully transparent and interactive process of engagement combined with reviewer identification exposes the level of engagement and the added value from each participant.\n\nCertification can be provided to referees based on their engagement with the process: community evaluation of their contributions (e.g. Amazon, Reddit, or Stack Exchange), combined with their reputation as authors. Rather than having anonymous or pseudonymous participants, for peer review to work well it would require full identification, to connect on-platform reputation and authorship history. Rather than a journal-based form, certification is granted based on continuing engagement with the research process and is revealed at the article (or object) and individual level. Communities would need to decide whether or not to set engagement filters based on quantitative measures of experience or reputation, and what this should be for different activities (e.g., as employed at ScienceOpen). This should be highly appealing not just to researchers, but also to those in charge of hiring, tenure, promotion, grant funding, and research assessment, and therefore could become an important factor in future policy development. Models like Stack Exchange are ideal candidates for such a system, because achievement of certification takes place via a process of community engagement and can be quantified through a simple and transparent up-voting and down-voting scheme, combined with achievement badges. Any outputs from assessment could be portable and applied to ORCID profiles, external webpages, and continuously updated and refined through further activity. While a star system does not seem appealing due to the inherent biases associated with it, this quantitative way of “reviewing the reviewers” creates a form of dynamic social reputation. As this is decoupled from journals, it alleviates all of the well-known issues with journal-based ranking systems and is fully transparent. By combining this with moderation, as outlined above, gaming can also be prevented (e.g., by providing numerous low quality engagements). Integrating a blockchain-based token system could also reduce potential for such gaming. Most importantly though, is that the research communities, and engagement within them, form the basis of certification, and reputation should evolve continuously with this.\n\nIncentives are required to motivate and encourage wider participation and engagement with peer review. As such, this requires lowering the threshold of entry for different research communities. The most widely-held reason for performing peer review is a sense of academic altruism or duty to the research community. However, at present this is imbalanced and researchers still receive far too little credit as a way of recognizing their efforts. This is directly tied to certification and reputation, as above, which is the ultimate goal of any incentive system.\n\nNew ways of incentivizing peer review can be developed by quantifying engagement with the process and tying this in to academic profiles, such as ORCID. To some extent this is already performed via Publons, where the records of individuals reviewing for a particular journal can be integrated into ORCID. This could easily be extended to include aspects from Reddit, Amazon, and Stack Exchange, where participants receive virtual rewards, such as points or karma, for engaging with peer review and having those activities further evaluated and ranked by the community. After a certain quantified threshold has been achieved, a hierarchical award system could be developed into this, and then be subsequently integrated into ORCID. Such awards or badges could include “Top reviewer”, “Verified reviewer”, “Community leader’,’ or whatever individual communities decide is best for them. This can form an incentive loop, where additional engagement abilities are acquired based on achievement of such badges.\n\nHighly-rated reviews gain more exposure and more credit, thus there incentive is to engage with the process in a way that is most beneficial to the community. Engagement with peer review and community evaluation of that then becomes part of a verified academic record, which can then be used as a way of establishing individual prestige. Such a system would be automatically integrated with any published content itself and objects could be similarly granted badges, such as “Community reviewed,” “Community accepted,” or “500 upvotes” as a way of quantifying the process. Therefore, there would be a dual incentive for authors to maximize engagement from the research community and for that community to productively engage with content. A potential extension of this in the form of monetization (e.g., through a blockchain protocol) is perhaps unwise, as it may lead to a distortion of incentives.\n\nNone of the ideas proposed here are particularly radical, representing more the recombination of existing variants that have succeeded or failed to varying degrees. A key challenge that our proposed hybrid system will have to overcome is simultaneous uptake across the whole scholarly ecosystem. In particular, this proposed system involves a requirement for standardised communication between a range of key participants. Real shifts will occur where elements of this system can be taken up by specific communities, but remain interoperable between them. Identifying sites where stepwise changes in practice are desirable to a community is an important next step. However, it is clear that recent advances in technology can play a significant role in systemic changes to peer review. High quality implementations of these ideas in systems that communities can choose to adopt may act as de facto standards that help to build towards consistent practice and adoption.\n\nOne aspect that we did not examine in detail is the use of instant messaging services, like Slack or Gitter. These are widely used for project communication and operate analogous to a real-time collaboration system with instantaneous and continuous “peer review”. While such activities can be used to supplement other hybrid platforms, as an independent or stand-alone mode of peer review the concept is quite distant from the other models that have been discussed here.\n\n\n5 Conclusions\n\nIf the current system of peer review were to undergo peer review, it would undoubtedly achieve a “revise and resubmit” decision. As Smith (2010) succinctly stated, “we have little or no evidence that peer review ‘works,’ but we have lots of evidence of its downside”. The Internet has changed our expectations of how communication works, and enabled a wide array of new, technologically-enabled possibilities to change how we communicate and interact online. Peer review has also recently become an online endeavor, but few organizations who conduct peer review have adopted Internet-style communication norms. This leaves a gap in what is possible with current technology and social norms and what we are doing to ensure the reliability and trustworthiness of published science. Peer review is a critical part of an effective scientific enterprise, but many of those who conduct peer review and depend upon it do not fully understand the theoretical and empirical basis for it. This means that our efforts to advance and change peer review are being driven by organizational goals such as market position and profit, and not by the needs of academia.\n\nExisting, popular online communication systems and platforms were designed to attract a huge following, not to ensure the ethics and reliability of effective peer review. Numerous front-end Web applications already implement all of the essential core traits for creating a widely distributed, diverse peer review ecosystem. We already have the technology we need. However, it will take a lot of work to integrate new technology-mediated communication norms into effective, widely-accepted peer review models, and connect these together seamlessly so that they become inter-operable as part of a sustainable scholarly communications infrastructure. Identity is a core factor driving online communication adoption and social norms and practices of current peer review – both how it is traditionally conducted with editorial management, and what will be possible with novel models online.\n\nThese socio-technological barriers cannot be overcome by simply creating platforms and expecting researchers to use them. Rather, as others have suggested (e.g., Moore et al. (2017); Prechelt et al. (2017)), platforms should be developed with community engagement, education, and capacity building as core traits, in order to help understand the cultural processes and needs of different disciplines and create solutions around those. Coordinated efforts are required to teach and market the purpose of peer review to researchers. More effective engagement is clearly required to emphasize the distinction between the idealized processes of peer review, along with the perceptions and applications of it, and the resulting products and services available to conduct it. This would help close the divergence between the social ideology and the technological application of peer review.\n\nIn this paper, we present an overview of what the key features of a hybrid, integrated peer review and publishing platform might be and how these could be combined. These features are embedded in research communities, which can not only set the rules of engagement but also form the judge, jury, and executioner for quality control, moderation, and certification. The major benefit of such a system is that peer review becomes an inherently social and community-led activity, decoupled from a traditional journal-based system, and instead becomes part of the commons. The “Principle of Maximum Bootstrapping” outlined by Kelty et al. (2008) is highly congruent with this social ideal for peer review, where new systems are based on existing communities of expertise, quality norms, and mechanisms for review. Diversifying peer review in such a manner is an intrinsic part of a system of reproducible research (Munafò et al., 2017). Making use of persistent identifiers such as DataCite, CrossRef, and ORCID will be essential in binding the social and technical aspects of this to an interoperable, sustainable and open scholarly infrastructure (Dappert et al., 2017).\n\nWe recognize that any technological advance is rarely innocent or unbiased, and while Web 2.0 technologies open up the possibility for increased participation in peer review, it would still not be inherently democratic (Elkhatib et al., 2015). As Belojevic et al. (2014) remark, when considering tying reputation engines to peer review, we must be aware that this comes with implications for values, norms, privilege and bias, and the industrialization of the process (Lee et al., 2013). Peer review is socially and culturally embedded in scholarly communities and has an inherent diversity in values and processes, which we must have a deep awareness of and appreciation for. Evidence-based research on peer review itself would help to build our collective understanding of the process and guide the design of ad-hoc solutions (Rennie, 2016). Further research should also focus on the challenges faced by researchers from peripheral nations, particularly for those who are non-native English speakers, and increase their influence as part of the globalization of research (Fukuzawa, 2017; Salager-Meyer, 2008, Salager-Meyer, 2014). The scholarly publishing industry could help to foster such research by starting to share its data on peer review (Squazzoni et al., 2017), with their incentive being to help improve the process.\n\nAcademics have been entrusted with an ethical imperative towards accurately generating, transforming, and disseminating new knowledge through peer review and scholarly communication. Peer review started out as a collegial discussion between authors and editors. Since this humble origin, it has vastly increased in complexity and become systematized and commercialized in line with the neo-liberal evolution of the modern research institute. This system is proving to be a vast drain upon human and technical resources, due to the increasingly unmanageable workload involved in scholarly publishing. There are lessons to be learned from the Open Access movement, which started as a set of principles by people with good intentions, but was subsequently converted into a messy system of mandates, policies, and increased costs that is becoming increasingly difficult to navigate. Commercialization has inhibited the progress of scholarly communication, and can no longer keep pace with the generation of new ideas in a digital world.\n\nThe research community has the opportunity to help create an efficient and socially-responsible system of peer review. The history, technology, and social justification to do so all exist. Research communities need to embrace the opportunities gifted to them and work together across stakeholder boundaries (e.g., with research funders, libraries and professional communicators) to create a more optimal system of peer review aligned with the diverse needs of non-independent research communities. By decoupling peer review, and with it scholarly communication, from commercial entities and journals, it is possible to return it to the core principles upon which it was founded more than a century ago. Through this, knowledge generation and access can become a democratic process again, and academics can fulfil the criteria that has been entrusted to them as creators and guardians of knowledge.", "appendix": "Competing interests\n\n\n\nJPT works for ScienceOpen; TRH works for OpenAIRE.\n\n\nGrant information\n\nTRH was supported by funding from the European Commission H2020 project OpenAIRE2020 (Grant agreement: 643410, Call: H2020-EINFRA-2014-1).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nCRM, DG, DM, DSK, DPOD, JNK, KEN, MP, MS, SK, SR, and YE thank those who posted on Twitter for making them aware of this project. During the writing of this manuscript, we also received numerous refinements, edits, suggestions, and comments from an enormous external community. DG has been supported by the Alexander von Humboldt (AvH) Foundation. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nWikipedia contributors: Digital medievalist. 2017; Accessed: 2017-6-16. Reference Source\n\nXiao L, Askin N: Wikipedia for academic publishing: advantages and challenges. Online Inform Rev. 2012; 36(3): 359–373. Publisher Full Text\n\nXiao L, Askin N: Academic opinions of Wikipedia and open access publishing. Online Inform Rev. 2014; 38(3): 332–347. Publisher Full Text\n\nYarkoni T: Designing next-generation platforms for evaluating scientific output: what scientists can learn from the social web. Front Comput Neurosci. 2012; 6: 72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nYli-Huumo J, Ko D, Choi S, et al.: Where Is Current Research on Blockchain Technology?-A Systematic Review. PLoS One. 2016; 11(10): e0163477. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nZamiska N: Nature cancels public reviews of scientific papers. Wall Str J. 2006; Date accessed: 2017-06-12. Reference Source" }	[ { "id": "24353", "date": "07 Aug 2017", "name": "David Moher", "expertise": [ "Reviewer Expertise Journalology (publication science)" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript is a herculean effort and enjoyable read. I learned lots, (and I think the paper will be a good resource for anybody interested in the field of peer review) which for me is usually a good sign of a paper’s worth.\n\nThe authors report on many aspects of peer review and devote considerable attention to some challenges in the field and the enormous innovation the field is witnessing.\n\nI think the paper can be improved: 1. It is missing a Methods section. It was unclear to me whether the authors conducted a systematic review or whether they used a snowballing technique (starting with seed articles) to identify the content discussed in the paper? Did the authors search electronic databases (and if so which ones?) What were their search strategies and/or did they rely on their own file drawers? Are all the peer review innovations/systems/approaches identified by the authors discussed or did they only discuss some (i.e., was a filter applied?)? With a focus on reproducibility I think the authors need to document their methods.\n\n2. I think the authors missed an important opportunity to discuss more deeply the need for evidence with all the current and emerging peer review systems (the authors reference Rennie 20161 in their conclusions. I think the evidence argument needs to be made more strongly in the body of the paper). I do not think the paper is strong enough regarding the large swaths of the peer review processes (current and innovations) for which there is no evidence2 and it is difficult to gain access to peer reviews to better understand their processes and effectiveness – open the black box of peer review3.\n\n3. There is limited data to inform us about several of the current peer review systems and innovations. In clinical medicine new drugs do not simply enter the market. They need undergo a rigorous series of evaluations, typically randomized trials prior to approval. Shouldn’t we expect something similar for peer review in the marketplace? It seems to me that any peer review process/innovation in development or released should have an evaluation (experimental, whenever possible) component integrated into it. Without evaluation we will miss the opportunity to generate data as to the effectiveness of the different peer review systems and processes. Research is central to getting a better understanding of peer review. It might useful for the authors to mention the existence of some groups/outlets committed to such research – PEERE (http://www.peere.org/) and the International Congress on Peer Review and Scientific Publication (http://www.peerreviewcongress.org/index.html). There is also a new journal committed to publishing peer review research (https://researchintegrityjournal.biomedcentral.com/).\n\n4. In section 1.3 of the paper the authors could add (or replace) Jefferson 20024 with Bruce5. The Bruce paper is also important for two additional reasons not adequately discussed in the paper: how to measure peer review and optimal designs for assessing the effects of peer review.\n\nConcerning measurement of peer review, there is accumulating evidence that there is little agreement as to how best to measure it. Unlike clinical medicine where there is a growing recognition of the need for core outcome set assessments (http://www.comet-initiative.org/) across all studies within specific content areas (e.g., atopic eczema/dermatitis clinical trials) we have not yet developed such an approach for peer review. Without a core outcome set for measuring peer review it will continue to be difficult to know what components of peer review researchers are trying to measure.\nSimilarly, without a core outcome set it will be difficult to aggregate estimates of peer review across studies (i.e., to do meaningful systematic reviews on peer review).\n\nConcerning the second point – there is little agreement as to an optimal design to evaluate the effectiveness of peer review. This is a critical issue to remedy in any effort to assess the effectiveness of peer review.\n\n5. The paper assumes (at least that’s how I’ve interpreted it – the paper is silent on this issue) that peer reviewers are all similarly proficient in peer reviewing. There is little training for peer reviewers (new efforts by some organizations such as Publons Academy are trying to remedy this). I started my peer-reviewing career without any training, as did many of my colleagues. If we do not train peer reviewers to a minimum globally accepted standard we will fail to make peer review better.\n\n6. Peer review does not function in a vacuum. The larger ecosystem includes other players’ most notably scientific editors. There is little discussion in the paper about this relationship and its potential (dys)function6.\n\n7. In section 2.2.1 you could also add at least one journal has an annual prize for peer reviewing (Journal of Clinical Epidemiology – JCE Reviewer Award: http://www.jclinepi.com/)\n\n8. In the competing interests section of the paper it indicates that the first author works at ScienceOpen although the affiliation given in the paper is Imperial College London. Is this a joint appointment? Clarification is needed. A similar clarification is required for TRH.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Yes\n\nAre all factual statements correct and adequately supported by citations? Yes\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [ { "c_id": "3005", "date": "12 Sep 2017", "name": "Xenia van Edig", "role": "Reader Comment", "response": "Comment on Table 2 I would like to point out that Atmospheric Chemistry and Physics [ACP] (only mentioned in table 2) was the first journal which applied a public review prior to publication, but that it is not the only journal which applies the so-called Interactive Public Peer Review. Besides ACP, 17 other journals published by Copernicus Publications apply this approach. In addition, there are other initiatives like the Economics e-journal or SciPost which apply similar approaches but are not affiliated with Copernicus. I would like to summarize my concerns/suggestions in the following: In the Interactive Public Peer Review authors’ manuscripts are posted as discussion papers (preprints), reviewer comments (i.e. reports) are posted alongside the manuscript (reviewers can decide whether they want to be anonymous or eponymous), and the scientific community is invited to comment on the manuscript prior to formal article publication. Most of the 17 journals applying this approach also publish the referee reports and other documents which were created after the discussion (peer-review completion) after the final acceptance of the manuscript as a journal article (e.g. https://www.biogeosciences.net/14/3239/2017/bg-14-3239-2017-discussion.html). The Interactive Public Peer Review and its development are described in https://www.atmospheric-chemistry-and-physics.net/pr_short_history_interactive_open_access_publishing_2001_2011.pdf http://journal.frontiersin.org/article/10.3389/fncom.2012.00033/full http://ebooks.iospress.nl/publication/42891 The approach is not reflected correctly in table 2: firstly, not only ACP is applying this approach. Secondly, it does not require a consensus decision. As in the traditional peer-review approach, the editor takes his/her decision based on the reviewer reports and other comments in the discussion (and if applicable request revisions after the discussion). The discussions as public peer review provide invited comments by referees, authors, and editors, as well as spontaneous comments by interested readers becoming part of the manuscript’s evolution. Therefore, the interactive journals of Copernicus Publications do not fit into the category “collaborative”. Following the types “pre-peer review commenting” and “post-publication commenting” of table 2, it could be named “pre-publication commenting”. The review type “pre-publication” in table 2 is misleading since it is the only one that is not transparent; therefore I suggest having at least the addition “pre-publication (closed)”. Otherwise, if you included my suggestion from 2 as “pre-publication commenting”, one could think that “pre-publication” and “pre-publication commenting” are as similar to “post-publication commenting” and “post-publication”, which is not the case. Both post-publication approaches are transparent, whereas only our pre-publication approach is." } ] }, { "id": "24355", "date": "14 Aug 2017", "name": "Virginia Barbour", "expertise": [ "Reviewer Expertise Editing", "Peer review", "Publication Ethics" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for asking me to review this paper.\n\nIronically, but perhaps not surprisingly, this was quite a hard paper to peer review and I don’t claim that this peer review does anything more than provide one (non-exhaustive) opinion on this paper. My views on peer review, which have formed over more than 15 years of being involved in editing and managing peer review will have coloured my peer review here.\n\nI think it's useful to regard all journal processes, which includes peer review, as components of the QC which begins with checking for basics such as the presence of ethics statements or trial registration, or the use of reporting guidelines for example, through to in depth methodological review. I don't think that any of the parts of the system of QC, including peer review, are perfect but the system is one component of attempting to ensure reproducibility, itself a core role of journals. The very basic functions of QC are often not given enough emphasis, though they are going to become more important as, for example, other types of publication such as preprints increase in popularity.\n\nGeneral Comments\n\nThis is a wide ranging, timely paper and will be a useful resource.\n\nMy main comment is that this is a mix of opinion, review, and thought experiment of future models. While all of these are needed in this area, for the review part of the paper, it would be much strengthened with a description of the methodology used for the review, including databases searched for information and keywords used to search, etc.\n\nThe paper is very long and there is a substantial amount of repetition. I think the introduction in particular could be much shortened - especially as it contains a lot of opinion, and repetition of issues dealt with elsewhere in the paper.\n\nThe language of the paper is also quite emotive in places and though I would personally agree with some of the sentiments I don't think they are helpful in making the authors’ case eg in Table 2 assessment of pre publication peer review is listed as Non-transparent,impossible to evaluate,biased, secretive, exclusive\n\nOr The entrenchment of the ubiquitously practiced and much more favored traditional model (which, as noted above, is also diverse) is ironically non-traditional, but nonetheless currently revered.\n\nI think it worth reviewing the language of the paper with that in mind.\n\nAlthough it arises in a number of places I don’t feel the authors address fully the complexity of interdisciplinary differences. The introduction would have been a good place to set this down.\nThere is no mention of initiatives such as EQUATOR which have been important in improving reporting of research and its peer review. http://www.equator-network.org/\n\nI was surprised to see very little discussion of the problems associated with commenting - especially of tone - that can arise on anonymous or pseudonymous sites such as Pubpeer and reddit.\n\nThere was no discussion of post publication reviews which originate in debates on twitter. There have been some notable examples of substantial peer review happening - or at least beginning there eg that on arsenic life1.\nThere are quite a few places where initiatives are mentioned but not referenced or hyperlinked. eg Self Journal of Science.\n\nSpecific comments\n\nIntroduction I would take issue with the term “gold standard”. In my view many of the issues arising from peer review are that it is held to a standard that was never intended for it.\n\nIntroduction paragraph 2 - where PLOS is mentioned here it should be replaced by PLOS ONE - the other journals from PLOS have other criteria for review. I am surprised that PLOS ONE does not get more of a mention in how much of a shift it represent in its model of uncoupling objective from subjective peer review, and how it led to the entire model for mega journals.\n\n1.1.1 “The purpose of developing peer reviewed journals became part of a process to deliver research to both generalist and specialist audiences, and improve the status of societies and fulfil their scholarly missions”\n\nI think it is worth noting that another function of peer review at journals was that it was part of earliest attempts of ensuring reproducibility - which is of course a very hot topic nowadays but in fact has its roots right back to when experiments were first described in journals.\n\n“From these early developments, the process of independent review of scientific reports by acknowledged experts gradually emerged. However, the review process was more similar to non-scholarly publishing, as the editors were the only ones to appraise manuscripts before printing”\n\nThere is a misconception here, which I think is quite common. In the vast majority of cases editors are also peers, and may well be “acknowledged experts” - in fact certainly will be at society journals. The distinction between editors and peer reviews can be a false one with regard to expertise.\n\n1.1.2 where publishers call upon external specialists to validate journal submissions.\n\nIt is important to note that it is editors who manage review processes. Publisher are largely responsible for the business processes; editors for the editorial processes.\n\nBy allowing the process of peer review to become managed by a hyper-competitive industry, developments in scholarly publishing have become strongly coupled to the transforming nature of academic research institutes. “ These have evolved into internationally competitive businesses that strive for quality through publisher-mediated journals by attempting to align these products with the academic ideal of research excellence (Moore et al., 2017)”\n\nI am not sure what is meant by “these” in this second sentence, nor what is meant by a “publisher-mediated journal”. Virtually all journals have a publisher - even small academic-led ones.\n\n1.1.3 This practice represents a significant shift, as public dissemination was decoupled from a traditional peer review process, resulting in increased visibility and citation rates (Davis & Fromerth, 2007; Moed, 2007).\n\nMany papers posted on arxiv.org do go on to be published in peer reviewed journals. Are these references referring to increased citation of the preprints or the version published in a peer reviewed journal?\n\nThe launch of Open Journal Systems (openjournalsystems.com; OJS) in 2001 offered a step towards bringing journals and peer review back to their community-led roots.\n\nThe jump here is odd. OJS actually can support a number of models of peer review, including a traditional model of peer review, just on a low cost open source platform, not a commercial one. The innovation here is the technology.\n\nDigital-born journals, such as PLOS ONE, introduced commenting on published papers.\n\nHere the reference should be to all of PLOS as commenting was not unique to PLOS ONE. However, the better example of commenting is the BMJ which had a vibrant paper letters page which it transformed very successfully to its rapid responses - and it remains the journal that has had most success http://www.bmj.com/rapid-responses.\n\nOther services, such as Publons, enable reviewers to claim recognition for their activities as referees.\nOriginally Academic Karma http://academickarma.org/ had a similar purpose though now it has a different model - facilitating peer review of preprints.\n\nFigure 2 PLOS ONE and ELife should be added to this timeline. Elife’s collaborative peer review model is very innovative. I am not sure why Wikipedia is in here.\n\n1.3 One consequence of this is that COPE, the Committee on Publication Ethics (publicationethics.org), was established in 1997 to address potential cases of abuse and misconduct during the publication process.\n\nCOPE was first established because of issues related to author misconduct which had been identified by editors. Though it does now have a number of cases relating to peer review , the guidelines for peer review came much later and peer review was not an early focus.\n\nTaken together, this should be extremely worrisome, especially given that traditional peer review is still viewed almost dogmatically as a gold standard for the publication of research results, and as the process which mediates knowledge dissemination to the public.\n\nI am not sure I would agree. Every person I know who works in publishing accepts that peer review is an imperfect system and that there is room for rethinking the process. Sense about Science puts it well in its guide: ”Just as a washing machine has a quality kite-mark, peer review is a kind of quality mark for science. It tells you that the research has been conducted and presented to a standard that other scientists accept. At the same time, it is not saying that the research is perfect (nor that a washing machine will never break down). http://senseaboutscience.org/wp-content/uploads/2016/09/peer-review-the-nuts-and-bolts.pdf\n\nTable 2. Note that quite a few of these approaches can co-exist. Under post publication commenting PLOS ONE should be PLOS. BMJ should be added here.\n\n1.4 Quite a lot of subscription journals do reward reviewers by providing free subscriptions to the journal - or OA journals provide discounts on APCs (including F1000). Furthermore, some reviewers are paid, especially statistical reviewers.\n\n2.2.2\n\nHence, this [Wiley] survey could represent a biased view of the actual situation.\n\nI’d like to see evidence to support this statement.\n\n2.2.3 The idea here is that by being able to standardize peer review activities, it becomes easier to describe, attribute, and therefore recognize and reward them\n\nI think the idea is to standardise the description of peer review, not the activity itself. Please clarify.\n\n2.4.2. Either way, there is little documented evidence that such retaliations actually occur either commonly or systematically. If they did, then publishers that employ this model such as Frontiers or BioMed Central would be under serious question, instead of thriving as they are.\n\nThis sentence seems to be in contradiction to the phrase below: In an ideal world, we would expect that strong, honest, and constructive feedback is well received by authors, no matter their career stage. Yet, it seems that this is not the case, or at least there seems to be the very real perception that it is not, and this is just as important from a social perspective. Retaliations to referees in such a negative manner represent serious cases of academic misconduct\n\n2.5.1. This process is mediated by ORCID for quality control, and CrossRef and Creative Commons licensing for appropriate recognition. They are essentially equivalent to community-mediated overlay journals, but with the difference that they also draw on additional sources beyond pre-prints.\n\nThis is an odd description. In what way does ORCID mediate for quality control?\n2.5.2 Two-stage peer review and Registered Reports.\n\nRegistration of clinical trials predated registered reports by a number of years and it would be useful to include clinical trial registration in this section.\n3 Potential future models\n\nNB I didn’t review this section in detail.\n\n3.5 as was originally the case with Open Access publishing,\n\nThe perception of low quality in OA was artificially perpetuated by traditional publishers more than anything else - it was not inherent to the process.\n3.5 Wikipedia and PLOS Computational Biology collaborated in a novel peer review experiment which would be worth mentioning - see http://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.10024462.\n3.9.2 Data peer review. This is a vast topic and there are many initiatives in this area, which are not really discussed at all. I would suggest this section should come out - especially as earlier on it is noted that the paper focuses mainly on peer review of traditional papers. I would also suggest taking out the parts on OER and books.\n\nIs the topic of the review discussed comprehensively in the context of the current literature? Partly\n\nAre all factual statements correct and adequately supported by citations? Partly\n\nIs the review written in accessible language? Yes\n\nAre the conclusions drawn appropriate in the context of the current research literature? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-1151
https://f1000research.com/articles/6-1003/v1	26 Jun 17	{ "type": "Research Article", "title": "Assessment of injection safety in Ha Dong General Hospital, Hanoi, in 2012", "authors": [ "Phan Van Tuong", "Tran Thi Minh Phuong", "Bui Thi My Anh", "Trang Huyen Thi Nguyen", "Phan Van Tuong", "Tran Thi Minh Phuong", "Bui Thi My Anh" ], "abstract": "Background: Injection is one of the most frequently used medical methods to introduce drugs or other substances into the body for purposes of treatment or prevention. Unsafe injection can cause adverse outcomes, such as abscess and anaphylactic shock, and increases the risk of blood-borne transmission of viruses to patients and health care workers, as well as the community. Recognizing the importance of injection safety, in 2000 the Vietnamese Ministry of Health (MOH) collaborated with the Vietnam Nurses Association to launch the “Safe injection” program throughout the country, including Hanoi. Methods: This cross-sectional study, combining quantitative and qualitative analysis, was conducted from February to August 2012 in Ha Dong General Hospital using a structured questionnaire and observation checklist. The target population of the study was 109 nurses working in clinical departments and 436 injections were observed. Results: The percentage of nurses who are familiar with injection safety standards was found to be 82.6%. The proportion of practical injections that met the 23 standards of injection safety set by the MOH amounted to 22.2%. The factors related to safe injection practice of nurses who are younger age group (OR=3.1; p<0.05) and lower amount of working years (OR=2.8; p<0.05). Conclusions: A low proportion of nurses performed correct safety injection practice, which raises the need for further training about this issue, especially among older nurses.", "keywords": [ "Injections", "safe injections", "injection safety" ], "content": "Introduction\n\nInjection plays an important role in medical treatment at hospitals and other medical institutions, especially those where many patients with serious health conditions are treated1. In terms of preventive medicine, vaccination has a significant impact on reducing the incidence and mortality of infectious diseases, which can be prevented by children’s vaccination2.\n\nDespite such positive outcomes, injection can also cause risk of abscess at the site of injection, nerve paralysis, allergic reaction, and anaphylaxis, and, in particular, the risks of transmission of blood-borne viruses to patients, healthcare workers (HCWs) and the community3,4. According to the World Health Organization (WHO), unsafe injection has become a very common issue and is practiced in many countries; it is the major cause of transmission of diseases such as hepatitis B, hepatitis C and HIV2,5,6. The WHO estimates that 50% of injections performed in developing countries are unsafe, and that as many as 20–80% of cases of hepatitis B virus infections are caused by unsafe injections2,5.\n\nIn Vietnam, realizing the importance of safe injection and the risks caused by unsafe injections, in 2000, the Ministry of Health, in collaboration with the Vietnam Nurses Association, launched and implemented the “Safe injection” program across the country7. However, results from some studies after this program was launched show that the rate of injections complying with adequate injection safety standards are not high enough, ranging from 6.0 to 22.6%7–12. This leading causes related to this low rate of safe injections are: nurses working in understaffed conditions, updated information on injection safety not being conveyed to nurses, non-compliance of technical procedures, and poor infection control operations in injection practices and sample handling, as well as poor management of sharp medical wastes13,14.\n\nHa Dong General Hospital, a level I hospital in Hanoi, with a capacity of 550 beds, including 33 departments and specialties, is responsible for the health care of people in the western part of Hanoi city. Following social development trends, the hospital always invests in quality improvement and advancement, including the \"Safe injection\" program launched by the Vietnam Nurses Association. To provide a description of the situation regarding injection safety in Ha Dong General Hospital, we have conducted a study with the following objectives: (1) Describing the status of injection safety in the hospital; (2) Describing the status of knowledge and injection safety practice of nurses working in the hospital; and (3) Identifying the factors related to nurses’ knowledge and safe injection practice.\n\n\nMethods\n\nA cross-sectional study was conducted from February to August 2012 in Ha Dong General Hospital, Hanoi.\n\nThe required sample size was calculated based on the WHO manual for sample size determination (http://apps.who.int/iris/handle/10665/40062). Applying the formula for calculating the one-ratio sample size where the expected rate of safe injections for Ha Dong General hospital was 51.2% (based on a previous study4), confidence level = 95%; and margin of error = 0.05; the minimum sample size was 384 injections. An additional sample size of 10% was added to the minimum sample size to avoid observation failure, resulting in the final sample size = 422 injections. All 109 nurses of the hospital were involved in the administering of injections. Therefore, the number of injections observed for each nurse was 422 /109 = 3.87, which was rounded up to 4 injections per nurse. Therefore, the total number of injections to be observed was 109 × 4 = 436 injections.\n\nThe selection of target objects for in-depth interviews and focus group discussions (Supplementary File 1): 2 in-depth interviews with leaders (Director and hospital Chief Nurse); 14 in-depth interviews with injection performing nurses (randomly recruited); 4 discussion focus groups with the participation of 4 to 6 chief nurses from treatment departments.\n\nFor quantitative research, we used a structured questionnaire with an observation checklist (Supplementary File 2 and Supplementary File 3) to collect data from 109 participants. Only one observer observed one nurse at one time. The observers were the chief nurses of this hospital. Meanwhile, we conducted in-depth interviews and focus group discussions about key topics, which included work intensity; equipment and instruments; financial factors; forms of reward and encouragement; risk and risk management in injection practices; and other factors affecting nurses’ practices of injection safety.\n\nData was encrypted, entered into Epidata 3.0 software, and analyzed using SPSS 16.0 software. Frequency and percentage were used to describe the quantitative data. Chi-squared was used to measure the differences between variables. Odd ratios were calculated to identify the factors associated with safety injection practice. Regarding qualitative data, content transcription from the in-depth interviews and focus discussion groups were categorized into the following topics: workload, equipment, financial factor, incentive and judgement; risk management in safety injection.\n\nThe study was approved by the IRB of Hanoi University of Public Health (No 029/2012/YTCC-HD3). Data collection procedures and the use of data for analysis were also approved by the directors of the Ha Dong General Hospital. Participants were asked to give written informed consent. They could withdraw from the study anytime without effects on their work or their benefits. Since we observed the regular tasks of the nurse, no informed consent was required from the patients.\n\n\nResults\n\nFigure 1 shows that 85.1% were intravenous injections; deep intramuscular injections accounted for 3.6%; and only 1.1% were subcutaneous injections, which were usually used for antibiotics testing.\n\nInjection rate in gluteal muscles accounted for 0.2% (Figure 2). In the in-depth interviews, where injection-performing nurses were interviewed about why gluteal muscles injections only accounted for 0.2% of the 3.6% of deep intramuscular injections, it was said that \"using deep intramuscular gluteal muscles causes less pain for the patient, but both patient and staff are reluctant to use this method due to cultural reasons\" (In-depth interview). As for the time of injection, among the 436 observations, the majority of injections were performed in the morning (62.6%) and 7.3% of injections performed in the evening; other injections were performed in the afternoon. In average, each patient received 3.1 injections.\n\nOf the total target population, 41 nurses, accounting for 37.6% of the target population, had been injured by sharp objects, including 36.6% who had been injured 2-3 times. It was mainly caused by performing the wrong injection procedure (75.6%), or due to the unexpected movement of the patient (17.1%), and negligence (7.3%). The majority of injuries were to the fingers, accounting for 97.6% of injuries. Regarding the time of day, most injuries happen in the morning (68.3%) followed by the evening (14.6%) and the afternoon (9.8%).\n\nOf the 109 nurses observed in the study, men accounted for 12.8%. The professional qualification of the majority of the nurses in this study was secondary level graduate (83.5%); the average age of nurses in the study was 38.4 ± 11.7 years. Among the overall nursing population, 75.2% were nurses, 22.9% were midwives, and only 1.8% were technicians. The proportion of young nurses working for 5 years or less accounted for 25.7%; 30.3% had more than 25 years of service.\n\nThe results in Table 1 shows that 91.7% received training on safe injection in the past year. Up to 26% received training twice in the past year. Most were trained in hospitals (75.2%), only 11% participated in training courses at the Provincial Health Offices, and 7.3% had never been trained in injection safety. In addition to the general training program, the chief nurse often provided guidance on safe injection practices and knowledge for the nurses at the hospital (98.2%). The majority of nurses (95.4%) knew that in treatment rooms of departments, materials on injection safety are readily available.\n\nTable 2 shows that the proportion of nurses having good knowledge (≥17/21 right answers; <17/21 right answers – insufficient knowledge) in injection safety was 82.6%, according to the questionnaire responses, but there were only 1 in 23 questions in which 100% nurses gave the correct response, of which content involved checking the quality of drugs before injection. There were 4 departments where some nurses gave 100% correct responses: emergency, ophthalmology, internal gastrointestinal, and internal cardiovascular departments. Nurses with a low rate of correct response were found at the ENT (66.7%) and Cardiovascular respiratory (66.7%) Departments, with the Odonto-stomatology Department having the lowest rate (25.0%). There were 11 questions with 90–90.9% rates of correct responses; four questions with rates of 80–80.9%. There were 3 questions that received the lowest rate of correct response: knowledge about practice to keep the gauze sterile at bleeding injection sites, 67.9%; proper disposal of sharps, 67.9%; and knowledge on limiting transmission of blood-borne diseases while giving injections, 65.1%.\n\nPractice in the preparation of materials and injection devices. The study results indicate that the preparation of injection instruments was relatively complete. 99.1% injections were fully prepared with anti-shock injection box on the trolley, 97.7% with sharp object containers and hand antiseptic in a convenient location on the injection trolley, 94.0% had sterile needles and syringes. Yet, 6% of injections were made when nurses had not checked the integrity of the needle packaging, and without injection trolley and equipment (2.1%). 86.5% of observed injections achieved all five main criteria.\n\nPractice of aseptic principles in the administration of injections. Table 3 shows that the rate of injections performed in which nurses had cleaned their hands before administration was 63.1%. In 17% of injections, needles remained on the bottles, and 20% of injections were performed when no antiseptic techniques were applied to the medication containers, or with unchecked needles. The rate of injections performed by nurses in compliance with 4 sterile criteria in the injection process was only 45.0%.\n\nPractice of safe injection techniques. In 97.9% of injections, the nurses identified the injection sites correctly, 83% of injections were performed in compliance with the 5 standard techniques (identify correct injection position, sterilize the skin before injection, check quality of drug, perform correct injection technique, and sterilize the skin after injection) and the rate of quality control of medicine was 85.1%. The rate of proper skin disinfection before injection was 91.1%, but only 81% complied with standard disinfection practice immediately after injection. The rate of injections complying with technical criteria of injection was 66.5%.\n\nInteractive communication with the patients. Via observation, the rate of injections in compliance with the 5-correct injection techniques (correct patient, correct drug, correct dose, correct injection way, correct time) was 100%, the rate of maintaining care records and medical order books was 93.3%, and the lowest rate was the communication and observation of patients while performing injections, especially interactive communication with patients after injection, which was only 67.7%. Results of in-depth interviews also showed that communication with patients while conducting injection was not sufficient; before injecting, most nurses performed observation, gave guidance and prepared for the injection, but communication during and after injection was given incompletely or superficially, \"their way of communication did not show any enthusiasm, or sympathetic and sharing attitude, and without motivation or encouragement to patients for their cooperation in the performance of injection\" - (Focus group discussion)\n\nPractice of prevention of infection risks for patients and the community. 46.1% of the injections were performed in compliance with all the four technical standards (wear glove when intravenous, did not use hands to remove the needle, isolate syringes and needles, hand wash after injection) to prevent risks for people receiving injections and the community. Similar to the rate of hand disinfection before injection, only 61.9% performed quick hand disinfection after injection. 68.1% wore hand gloves when administering intravenous injection. It was reported by nurses in in-depth interviews that “it is difficult to perform intravenous injection if gloves are worn, especially in providing injections to small children\"- (HCWs-PVS). The rate of nurses using their bare hands in covering and removing needle caps was 88.8%. The highest rate was the rate of injections in compliance with the provision of isolating needles immediately after injection, which was 93.3%.\n\nPractical injections meeting safety standards criteria. The rates of injections that meet the safety injection (SI) criteria (correct preparation of injection equipment, ensure sterility requirement, correct injection technique, correct communication with patients, prevent risk for people receiving injection and ensure the safety standard of injection) at different departments ranged from 11.1% to 33.3%, with the lowest in the Emergency Department, with only 4 in 36 injections (8.3%), the rate in the Odonto-stomatology Department was 12.5%, and the highest was in the Pediatrics Department, with 14/42 injections, accounting for 33.3%.\n\nTable 4 shows that there are six nurses who do not have any of the four observed injections in compliance with the SI standards (5.5%). Only 9/109 nurses had all four observed injections meeting the criteria of injection safety (8.3%). There were 26 nurses who have at least three injections meeting the required standards (26.9%).\n\nFactors related to safe injection. The rate of nurses with knowledge of SI in the nurses aged up to 30 years was 93.2%, 3.3 times higher than those aged over 30 years (75.4%) (OR = 4.4; p <0.05). No statically significant difference was found in the rate of nurses having knowledge of good SI between nurses of different genders and different levels of education (p> 0.05).\n\nAmong nurses with sufficient SI knowledge, the number of nurses with <10 years of work was 4.9 times higher than that of nurses with work experience of >10 years (OR = 4.9; p <0.05). The rate of nurses with sufficient SI knowledge among nurses who received training in the past year was 86.1%, which was 10.3 times higher than the untrained group. This difference was statistically significant (p <0.001). However, there was no statically significant difference in the rates of sufficient knowledge of SI and the different levels of nurses (for example: college nurses or university nurses) (p>0.05).\n\nThe rate of injury due to sharp objects during injections in the group with no knowledge of SI was 63.2%, which is 3.6 times higher compared to the group with knowledge about SI (32.2%). The difference was statistically significant (X2 = 6.39; p <0.05).\n\nFactors related to safe injection practice. Age was a statistically significant factor with regard to safe injection practices (X2 = 6.3, p <0.05), the rate of correct practice of SI in nurses <30 years was found to be 3.1 times higher than those >30 years old.\n\nTable 5 shows the percentage of correct practices in the nursing team with work experience of <10 years (32.7%); 2.5 times higher than the senior group (work experience >10 years) (14.8%). This difference was statistically significant (p <0.05). Professional qualifications and the number of injections / day of each nurse had no statically significant association with SI practice (p> 0.05). The rate of correct practice of the group with knowledge about SI (26.7%) was three times higher than the group with no knowledge (10.5%); this difference was not statistically significant (OR = 3, 09; p> 0.05).\n\nOf the 436 observed injections, 273 injections were observed in the morning (62.6%). Safe injection rate in the morning was 22%. The rate of injection safety at midday was the highest (88.9%); in the afternoon, 95 injections were observed, but no injections were found to meet all the 23 criteria of SI. There were statically significant differences regarding injection safety depending on the time of the day at which injections are applied, which the highest percentage of safety injection was at noon (X2 = 120.4; p <0.001).\n\nIntravenous injections were observed at the highest rate (62.4%), but only 19.1% were found to be meeting SI standards. Safe injection rate of the observed subcutaneous injections was 57.1%, and for injection in the skin, this was 33.3%. The difference in the rate of safe injection according to injection type was statistically significant (X2 = 23.4; p <0.001). The number of intravenous injections was the most directly observed, with 173 injections out of 436 injections observed (50.5%), but the safety injection rate was only 21.4%. The safe injection rate was lowest in the intravenous injections via fork / rubber joints (9.3%) and the highest intramuscular injections in the thigh quadriceps (60%). However, this difference was not statistically significant (p> 0.05).\n\nThe first observed injections had the highest safety rate of 58.7%. Safe injection rate of the observed second injections was 7.3%, 3rd was 10.0% and the fourth was 13.0%. This difference was statistically significant; (X2 = 112.7; p<0.001).\n\nThe results of the in-depth interviews show the cause of unsafe injection. One reason was mentioned is that nurses were overloaded their work: \"In the morning I have to injecting [sic] dozens of patients, so how can [I] follow the process!\". With this workload, they felt stressful and therefore, they could not follow the procedure of safe injection practice. Additionally, some nurses injected as their habits with old procedures, which did not ensure the safe injection practice: \"Nurses with high age are very fluent in the use of fluids but often follow the old procedures, often bypassing, cutting down the process, changing the way they are\" (In-depth interview). Another reason also mentioned is the regular supervision of the chief nurse “If the chief nurse regular[ly] supervises the injection procedure, the nurses will mandatorily follow the procedure of safe injection practice”.\n\n\nDiscussion\n\nThis study provided baseline evidence for further interventions to improve safe injection practice in Vietnam. This research showed that on average each patient received 3.1 injections. Compared to the results study in other countries, this rate is lower than result study of HAURI Global 2000 study15. However, this result is higher than Tu's study16,17 and research by the Vietnam Nurses Association in 201013,18,19. About 37.6% nurses had been injured by sharp objects. The sharp injury rate at Ha Dong Hospital is higher than the results of Muc's study13, Nguyen Tu's 2005 study16, and lower than that of the Vong et al’s study in Cambodia20. Most of the injuries occurred in the morning. Unintended activities is the cause of most injuries. Meanwhile, sharp instruments injuries accounted for most of the fingers wounds\n\nKnowledge about safe injection standards of nurses. The ratio of nurses having knowledge about safe injection was found to be higher in the present study compared with the study of Ernest et al. at City Hospital Benin Nigeria21. These rates are lower than those of Phan Canh Chuong at the Hue Central Hospital9, but higher than that found in a previous study by Thanh7.\n\nNearly half of injections followed the 4 sterile standards. For example, 50% of injections followed regulation on communication standards, and most injections followed proper safety standards for injected persons; however, 17% and 32% of injections did not isolate the needle and syringe immediately after injection and only 32% used gloves when injecting intravenously.\n\nNurses aged up to 30 years had better knowledge and higher rate of safe injection practices than nurses >30 years old. Nurses with less than 10 years of work experience had better knowledge level and higher safe injection practices than senior nurses with 10 years or more experience. Regarding training, nurses trained for 1 year had better knowledge than untrained groups, and as a result untrained groups were more likely to be exposed to accidental injuries than that of knowledgeable groups. These findings were similar to other previous studies8–10. The rate of correct practice of the group with sufficient knowledge was higher than the group with insufficient knowledge, but this difference was not statistically significant (p > 0.05), which was consistent with other studies7,16,19. The results of the in-depth interviews also showed that old habits, e.g. bypassing the injection process, and the supervisor's supervision were also influential. Meanwhile, injection timing, parenteral administration and order injections were observed factors that have statistically significant relationship (p < 0.05) with the rate of safe injections of the hospital.\n\nA number of recommendations can be made based on the results of the study: (1) Enhancing the sterilization performance, reducing the risk of infection due to injury; (2) Promoting training courses to improve knowledge and skills, educational communication to increase knowledge and awareness of risk of injections; (3) Establishing a regular injection safety monitoring and assessment program - the results and related information must be reported to management and disseminated to hospital staff; (4) Enhancing the inspection and supervision of regimes of reward and sanction, of emulation and commendation, and conducting research of SI assessment; (5) Focusing on the principles of sterilization, hand hygiene before injection, sterilization routine when taking drugs, and sterilization of needles in injection safety training, as well as on enhancing communication skills in dealing with patients.\n\n\nConclusion\n\nThis study found a low proportion of nurses performing correct safety injection practice, which raises the need for further training about this issue, especially among older nurses.\n\n\nData availability\n\nDataset 1: Raw data obtained from the questionnaire assessing knowledge for safe injection practice among nurses. doi, 10.5256/f1000research.11399.d16539022\n\nDataset 2: Raw data obtained from the observation assessing practice for safe injection practice among nurses. doi, 10.5256/f1000research.11399.d16539123\n\nThe transcripts of the in-depth-interview and focus group discussion are not available due to the sensitive information contained. However, this information will be made available for university researchers who send a request to Prof. Tuong Van Pham, PI of the study: pvt@huph.edu.vn.", "appendix": "Author contributions\n\n\n\nPVT, TTMP, BTMA, THTN was responsible for the research methods and prepared the first draft of the manuscript. PVT, TTMP, BTMA, THTN contributed to the experimental design and preparation of the manuscript. All authors were involved in the revision of the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nSupplementary material\n\nSupplementary File 1: In-depth interview guide and group discussion guide.\n\nClick here to access the data.\n\nSupplementary File 2: Questionnaire assessing knowledge for safe injection practice among nurses.\n\nClick here to access the data.\n\nSupplementary File 3: Observation checklist assessing practice for safe injection practice among nurses.\n\nClick here to access the data.\n\n\nReferences\n\nAssociation HN: Training document. 2010.\n\nHealth WatMo: Workshop materials engineering consultants national guidelines on injection safety. 2008.\n\nNaik A, Gharat V, Bansal RK: An assessment of injection practice in urban health centers of surat city: Are the health care workers safe. Reference Source\n\nYan YW, Yan J, Zhang GP, et al.: Prevalence of injections and knowledge of safe injections among rural residents in Central China. Singapore Med J. 2007; 48(8): 769–74. PubMed Abstract\n\nWHO Dept of Vaccines and Biologicals, WHO Dept of Protection of the Human Environment: First, do no harm” - Introducing auto-disable syringes and ensuring injection safety in immunization systems of developing countries. 2002. Reference Source\n\nYan Y, Zhang G, Chen Y, et al.: Study on the injection practices of health facilities in Jingzhou district, Hubei, China. Indian J Med Sci. 2006; 60(10): 407–16. PubMed Abstract \| Publisher Full Text\n\nThanh Đ: Result of safe injection at 13 hospitals selected by nursing association Hanoi 2010. Vietnam nursing association, 2010.\n\nCham TTM: Assessment of injection safety in Thanh Nhan district hospital in 2010. 2010.\n\nChuong PC: Assessment of injection safety in Hue Central Hospital in 2010. 2010.\n\nDung PT: Assessment of injection safety in Viet Duc Hospital in 2009. 2009.\n\nAnh PT: Assessment of injection safety in the Central Traditional Hospital in 2009. 2009.\n\nYen ĐH: Survey injection safety situation at the Hanoi Heart Hospital. 2011.\n\nMuc PD: Assess knowledge of injection safety and frequency of risk due to sharp objects for nursing-midwives in 8 provinces represent the first six months of 2005. Proceedings of the scientific research nationwide nursing; 2nd the Vietnam Nurses Association, 2005; 224–232.\n\nLinh NTM: Study on safe injection of nursing student at Obstetric Hospital Tien Giang province in 2008. 2008.\n\nHauri AM, Armstrong GL, Hutin YJ: The global burden of disease attributable to contaminated injections given in health care settings. Int J STD AIDS. 2004; 15(1): 7–16. PubMed Abstract \| Publisher Full Text\n\nTu NTN: Situation of safety in Binh Dinh province after 5 years in response to the campaign. National research project on nursing science, 2005.\n\nWHO HM: Training materials Safety Injection. 2004.\n\nTam NM: Results of the safe injections in hospital in Hanoi area. Proceedings of the research on nursing science - the first national conference on nursing science. 2001; 141–154.\n\nThanh Đ: Evaluate safety injection in 8 representative provinces, 2005. Proceedings of the second national research project on nursing science, 2005; 217–223.\n\nVong S, Perz JF, Sok S, et al.: Rapid assessment of injection practices in Cambodia, 2002. BMC Public Health. 2005; 5: 56. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nErnest SK: Injection safety: knowledge and practice among health workers. West Afr J Med. 2002; 21(1): 70–3. PubMed Abstract\n\nVan Tuong P, Phuong TTM, Anh BTM, et al.: Dataset 1 in: Assessment of injection safety in Ha Dong General Hospital, Hanoi, in 2012. F1000Research. 2017. Data Source\n\nVan Tuong P, Phuong TTM, Anh BTM, et al.: Dataset 2 in: Assessment of injection safety in Ha Dong General Hospital, Hanoi, in 2012. F1000Research. 2017. Data Source" }	[ { "id": "24673", "date": "14 Sep 2017", "name": "Mattias Larsson", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for a well written article with an important message - that injection safety can be improved which might decrease injection related disease transmission.\nComments: In Abstract it states:\nThe nurses who are familiar with injection safety standards ... 82.6%\n\nThe proportion of injections that met the 23 standards of injection safety ... 22.2%.\n\nA low proportion of nurses performed correct safety injection...\nThis indicates a large difference between knowledge and practice. It would also be of use to have the average of the number of safety standards that was recorded.\n\nIn Table 1. the headline \"Training courses on safe injection in the past year \" occurs twice with different figures which is not clearly explained in the text.\n\nExperience might not always guarantee better practice - \"rate of correct practice of SI in nurses <30 years was found to be 3.1 times higher than those >30 years old\"\nClarifications:\nQuestion \"Are all the source data underlying the results available to ensure full reproducibility?\"\nPartly as it would be good to have a shortlist of all 23 standards and how each of them was assessed as well as the result (average, SD, median)\n\nQuestion \"Are the conclusions drawn adequately supported by the results?\"\nPartly as there would be good to see a discussion regarding the large gap between knowledge and practice - why do people not behave as they know they should?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Partly\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3036", "date": "19 Sep 2017", "name": "Trang Nguyen", "role": "Author Response", "response": "Thank you very much for your feedback. We have added more details in conclusion of abstract and main text, which referred to the high level of knowledge but low level of practice; and the experience might not always guarantee better practice. We have also removed a part of table 1 that were duplications." } ] } ]	1	https://f1000research.com/articles/6-1003
https://f1000research.com/articles/6-2046/v1	23 Nov 17	{ "type": "Software Tool Article", "title": "miRcomp-Shiny: Interactive assessment of qPCR-based microRNA quantification and quality control algorithms", "authors": [ "Lauren Kemperman", "Matthew N. McCall", "Lauren Kemperman" ], "abstract": "The miRcomp-Shiny web application allows interactive performance assessments and comparisons of qPCR-based microRNA expression and quality estimation methods using a benchmark data set. This work is motivated by two distinct use cases: (1) selection of methodology and quality thresholds for use analyzing one's own data, and (2) comparison of novel expression estimation algorithms with currently-available methodology. The miRcomp-Shiny application is implemented in the R/Shiny language and can be installed on any operating system on which R can be installed. It is made freely available as part of the miRcomp package (version 1.3.3 and later) available through the Bioconductor project at: http://bioconductor.org/packages/miRcomp. The web application is hosted at https://laurenkemperman.shinyapps.io/mircomp/. A detailed description of how to use the web application is available at: http://lkemperm.github.io/miRcomp_shiny_app", "keywords": [ "microRNA", "miRcomp", "qPCR", "benchmark data" ], "content": "Introduction\n\nQuantitative real-time PCR (qPCR) is one of the most widely used methods to measure the expression of a target transcript. A variety of algorithms have been developed to estimate expression from qPCR fluorescence measurements. The vast majority of these algorithms were developed and tested using gene expression data1–3; however, they are now routinely applied to qPCR-based microRNA expression measurements. To evaluate the performance of these methods on microRNA data, we developed a benchmark data set and a collection of statistical assessments4. These and other recent assessments5 highlight the need to develop qPCR quantification and quality assessment methodology specifically tailored to microRNA expression platforms.\n\nCurrent methods to estimate expression from raw qPCR amplification data have been developed in a variety of programming languages (e.g. R, Python, SAS) and may be restricted to a particular operating system (e.g. Windows, Mac OS, Unix/Linux)6. Furthermore, these algorithms often return different data structures, complicating comparisons between methods. The miRcomp-Shiny web application provides a unified assessment environment that is platform independent and takes simple expression and quality matrices as input. This approach removes barriers to usage and facilitates the comparison of methods.\n\n\nMethods\n\nWe have developed a Shiny (http://shiny.rstudio.com/) interface to the miRcomp R package (version ≥ 1.3.3). Currently, six of the most widely-used algorithms to estimate miRNA expression and sample quality are included in the miRcomp-Shiny app. Each method provides both expression estimates and quality metrics. Assessments can be performed on individual algorithms, or two available algorithms can be compared. Researchers can also upload the results from their own method to be assessed. As new methods are developed and tested, we will continue to add these methods to miRcomp-Shiny. The development of a repository of qPCR-based miRNA expression estimation algorithms will be a valuable resource for researchers seeking to develop new methodology or comparison existing algorithms across a wide variety of assessment criteria.\n\nThe web application framework in R (Shiny) has enabled us to make several aspects of the miRcomp package more interactive than they were previously and facilitate comparisons that would have been difficult to make in R. Below we describe two common use cases that motivated the development of miRcomp-Shiny.\n\nWhen selecting methodology to analyze a data set, miRcomp-Shiny can be used to evaluate the performance of existing methods based on the benchmark data. The results of these evaluations can be used to guide the selection of an expression estimation algorithm and quality threshold based on the assessments most relevant to the user’s experiment. Additionally, one can examine the effect of changing quality thresholds on the performance of each method. The result of changes in the quality threshold are then displayed immediately for each assessment. This is particularly useful when selecting a quality threshold for one’s own data.\n\nAnother use case is comparison of a new method to an existing method. By providing current methods for comparison, researchers do not have to implement these algorithms themselves, which is often a substantial bottleneck in the development and assessment of novel algorithms. Additionally, we will continue to add new methods to miRcomp-Shiny. This will produce a richer set of available methods in a single location to guide comparisons. The success of this approach has been demonstrated by the affycomp webtool7,8.\n\nInstallation. To access miRcomp-Shiny locally, the miRcomp R package and all required dependencies can be installed from Bioconductor with the following commands:\n\nsource(\"http://bioconductor.org/biocLite.R\")\n\nbiocLite(\"miRcomp\")\n\nTo access miRcomp-Shiny remotely, simply go to:\n\nhttps://laurenkemperman.shinyapps.io/mircomp/\n\nInput. The miRcomp-Shiny app takes one or two quantification methods as input. These methods can be selected from the drop-down menus on the left panel (Figure 1). Alternatively, the user can upload the results of their own method by selecting the custom option from the menu. If the custom option is selected, the user is prompted to upload a matrix of quality values (qc) and a matrix of expression estimates (ct). Once the method or methods have been selected, each assessment plot contains additional assessment-specific options below the plotting window (Figure 1).\n\nThe results of several methods are available or the user can upload the results of their own method (as shown above). After selecting one or more methods, the user can examine the 5 assessment tabs: limit of detection, accuracy, precision, quality assessment, and titration response. Each tab has its own options shown at the bottom of the pane.\n\nOutput. The miRcomp-Shiny app produces five plots to assess the performance of the method or methods selected: Limit of Detection, Accuracy, Precision, Quality Assessment, and Titration Response.\n\n\nUse cases\n\nThe assessments performed by miRcomp-Shiny are based on a benchmark data set available at:\n\nhttp://bioconductor.org/packages/miRcompData/\n\nUsers wishing to assess quantification and quality control metrics beyond those currently implemented, can run any algorithm on those data and upload the resulting matrices of quality values (qc) and expression estimates (ct). Examples of these matrices are included as R data objects in the miRcomp package for each of the currently implemented methods.\n\n\nSummary\n\nThe success of the miRcomp Shiny web application and software will depend on new methods being developed and the application being used to test them. We have already begun encouraging people to use the package, and hope that readers will do the same. Widespread use of these tools will lead to improvements across all benchmarks in microRNA expression estimation, and the accessibility of the web application makes that possible.\n\n\nSoftware availability\n\nSoftware available from: https://laurenkemperman.shinyapps.io/mircomp/\n\nSource code available from: http://bioconductor.org/packages/miRcomp\n\nArchived source code as at time of publication: http://doi.org/10.5281/zenodo.10490749\n\nSoftware license: GPL-3", "appendix": "Author contributions\n\n\n\nL.K. developed the software under the supervision of M.N.M. All authors wrote and approved the final manuscript.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by the National Institutes of Health grant to M.N.M (R00-HG006853).\n\nThe authors confirm that the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nWe thank Dr. Marc Halushka for his helpful comments on this manuscript. The OpenArray chips were run by the staff at the Genetic Resources Core Facility, Johns Hopkins Institute of Genetic Medicine, Baltimore, MD.\n\n\nReferences\n\nRitz C, Spiess AN: qpcR: an R package for sigmoidal model selection in quantitative real-time polymerase chain reaction analysis. Bioinformatics. 2008; 24(13): 1549–1551. PubMed Abstract \| Publisher Full Text\n\nLievens A, Van Aelst S, Van den Bulcke M, et al.: Enhanced analysis of real-time PCR data by using a variable efficiency model: FPK-PCR. Nucleic Acids Res. 2012; 40(2): e10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRödiger S, Burdukiewicz M, Schierack P: chipPCR: an R package to pre-process raw data of amplification curves. Bioinformatics. 2015; 31(17): 2900–2. PubMed Abstract \| Publisher Full Text\n\nMcCall MN, Baras AS, Crits-Christoph A: A benchmark for microrna quantification algorithms using the openarray platform. BMC bioinformatics. 2016; 17(1): 138. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMestdagh P, Hartmann N, Baeriswyl L, et al.: Evaluation of quantitative miRNA expression platforms in the microRNA quality control (miRQC) study. Nat Methods. 2014; 11(8): 809–815. PubMed Abstract \| Publisher Full Text\n\nPabinger S, Rödiger S, Kriegner A, et al.: A survey of tools for the analysis of quantitative PCR (qPCR) data. Biomol Detect Quantif. 2014; 1(1): 23–33. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nCope LM, Irizarry RA, Jaffee HA, et al.: A benchmark for affymetrix genechip expression measures. Bioinformatics. 2004; 20(3): 323–331. PubMed Abstract \| Publisher Full Text\n\nIrizarry RA, Wu Z, Jaffee HA: Comparison of affymetrix genechip expression measures. Bioinformatics. 2006; 22(7): 789–794. PubMed Abstract \| Publisher Full Text\n\nKemperman L, McCall MN: mccallm/miRcomp-shiny: F1000Research Version (Version v1.9.0-F1000). Zenodo. 2017. Data Source" }	[ { "id": "28320", "date": "04 Dec 2017", "name": "Levi Waldron", "expertise": [ "Reviewer Expertise biostatistics", "metagenomics", "microbiome", "software for multi-omics data analysis" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nKemperman and McCall present a Shiny App that is a straightforward extension of their Bioconductor mirComp package, creating the plots provided by the package with sliders for the options of the plotting functions, using a suite of microRNA qPCR test data.\n\nMajor\n\nThere are some shortcomings in the app that initially made me think it would still be necessary to use the Bioconductor version directly: * I couldn’t find documentation or an option in the app on how to use one’s own data with the app (use case 1 from abstract). update I found the “custom” method option, but only by trial and error – this option should be emphasized within the app * I also couldn’t see documentation on how to add a novel algorithm for comparison (use case 2 from abstract). update I think I understand now that a novel algorithm would be applied prior to uploading the data to the app, but please clarify this in the manuscript and app * The figures don’t have axis labels or captions, so one has to look up the miRcomp vignette and reference manual to understand them\n\nIt would be helpful to state in the Introduction and on the web app page who the intended users of the app are, and who it’s not intended for. It would also be helpful to state in the Introduction how the “simple expression and quality matrices as input” would normally be generated, with specific instructions both for generating (e.g. pointing to documentation for the methods already in the tool and how data from added methods should be formatted), and for uploading to the tool. This should also be coupled with explicitly stating that the tool does not perform normalization of miR expression data (or, better yet, incorporating normalization into the tool), and that the tool only assesses already normalized miR expression data. The point of this comment is to make it clearer up front to a reader whether or not the tool is for them.\n\nAs the app takes simple expression and quality matrices as input, it would be helpful to point to instructions on how to prepare these matrices. It would seem that this requires using the R/Bioc command line, so the app may facilitate the comparison of methods but not remove barriers to usage. Again, it should just be clear up front what requirements to the user are for the intended use cases.\n\nThis may be outside the scope of the paper, but the tool would be of greater use to wet lab biologists if they could upload raw data, do the comparisons provided by the app, then download normalized data. This would probably significantly expand the number of potential users.\n\nI understand from the introduction that the tool intends to expand on methods for testing qPCR normalization used for miR expression data. But it would be helpful to state how the app is actually specific to miR expression data – is it just that it provides miR datasets for benchmarks? Or are some of the normalization methods miR-specific?\n\nWhen trying out the app at https://laurenkemperman.shinyapps.io/mircomp/, I constantly got the message “Disconnected from the server. Reload” I had to run the app locally to test it usefully. The authors may need an upgraded shinyapps.io account to support public usage. When reloading, all changes made to the settings are reset.\n\nMinor\n\nWith qpcRb4 as the first method, I get an error “need finite ‘ylim’ values”. I haven’t checked through all the plotting combinations.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "28323", "date": "18 Dec 2017", "name": "Simina M. Boca", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nI applaud Kemperman and McCall on the work they put forward to make methods comparisons more accessible to researchers interested in microRNA quantification. I generally agree with Dr. Waldron's comments. Specific points where I have reservations include:\n1) I am still not sure how users can add their own data to compare various methods on it. It seems like if one selects \"custom\" for one of the two methods, then one must load a processed version of the same dataset in order to compare a new algorithm to existing algorithms.\n2) In general, I think Dr. Waldron's comment on whether users still need to use the Bioconductor package directly is very valuable. It partly depends on what users the authors have in mind for the shiny app in terms of their level of R/bioinformatics expertise.\nTwo specific examples here:\na) If an unfamiliar user were to use this app, it seems like they would still need to go to the Bioconductor package to get the dataset. Perhaps the authors could include this dataset in the app and make it easier to download as both a CSV file and an .RData file? It would also be worthwhile to include a citation to the dataset, as opposed to needing to go through Bioconductor.\nb) More explanations need to accompany the plots and explain exactly what is being plotted and why - each plot should at least include the same type of level of detail used when writing a figure caption in a scientific journal. A more detailed introduction should also be provided, along with links to the Bioconductor package(s) and to this paper, but written so that it is at least somewhat self-contained. For example, the first plot, \"Limit of detection\" appears to not be a comparison plot at all, but rather to show just the limit of detection for the first method. What should the user expect to see here for a \"good\" method? What does \"Proportion Poor Quality\" even mean (is there a threshold that can be changed to indicate this, why is there never a boxplot for the proportion = 1?) For \"Accuracy\" it is also not clear what \"percentage of data to exclude\" means (why is it being excluded? quality issues?) For \"Accuracy\" and \"Precision,\" the Low/Medium/High values on the x-axis should be described, along with stating that within each category, the methods are being compared (maybe some dashed vertical lines between categories would also help here). In general, it should be indicated what one should look for in terms of one method having better performance compared to another.\nI strongly encourage the authors to make these changes/additions in order to allow a larger number of individuals to use and benefit from their tool.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? No\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] }, { "id": "28321", "date": "18 Dec 2017", "name": "Elana J. Fertig", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper presents a new Shiny application for analyses of qPCR data to facilitate comparison of methods. This software is important for the community and well suited to publication in F1000Research.\n\nMajor comments\n\nSome details of the software are hard to follow from the manuscript below (e.g., which methods are implemented, what benchmark datasets, etc). These are detailed in minor comments below, and must be fixed for readability of the manuscript. The authors may wish to add a table listing each of the methods and the quality thresholds that they yield. Use cases should report results from applying the method, not merely the datasets used for analysis. The manuscript should describe the range of possible analyses that can be performed with miRcomp-Shiny, expanding upon the “output section”. The annotations and help on the software available from https://laurenkemperman.shinyapps.io/mircomp/ require improvement for greater usability. Some examples are listed below:\nDataset descriptions describes the methods employed, but does not indicate which datasets are used. The platform seems limited to analysis of the miRcomp data. The software does not appear to enable input of new datasets which would be critical to its utility. The format of files for qc and ct elements for custom analyses are not specified. There is no ability to export processed datasets and/or assess the quality of specific miRNAs from the preprocessing implemented in this application.\n\nMinor comments\n\nThe Implementation subsection of the Methods should expand the sentence “Currently, six of the most widely-used algorithms to estimate miRNA expression and sample quality are included in the miRcomp-Shiny app” to clarify precisely which six algorithms are implemented and include citations to those methods. It should also clarify whether these 6 algorithms are representative of all in the miRcomp R package or a subset of the methods implemented in that package. “The benchmark data” referenced in the Methodology and quality selection threshold should be defined. Which datasets are included as benchmarks? How are they selected? It is unclear what variables the “quality thresholds” in the Methodology and quality section threshold section reference. The subsection “Comparison of novel algorithms…” should edit the sentence “Another use case is comparison of a new method to an existing method.” to read “Another use case is comparison of a data table with results from a new method to the existing methods implemented in the miRcomp-Shiny app.” The sentence “We have already begun encouraging people to use the package, and hope that readers will do the same.” Should be cut. The summary should place this tool in context of others in the literature and discuss its limitations / future work.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? No\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-2046
https://f1000research.com/articles/6-2036/v1	21 Nov 17	{ "type": "Research Article", "title": "Increased static postural sway after energy drink consumption: A randomized trial", "authors": [ "Martin G. Rosario", "Henry Collazo", "Milagros Mateo", "Maryví Gonzalez-Sola", "Flavia Bayron", "Henry Collazo", "Milagros Mateo", "Maryví Gonzalez-Sola", "Flavia Bayron" ], "abstract": "Background: Energy drinks consumption continues to grow since its appearance in the United States in 1997. Available evidence indicates that caffeine, their main ingredient, can alter the central nervous system (CNS). However, it is unknown how energy drinks alter the CNS postural control mechanism. The purpose of this study was to investigate how energy drinks can affect postural control after sensory perturbations during stance. Methods: 20 healthy adults, (11 males; 9 females) averaging 26.1 years of age, stood on a MatScan™ pressure mat, which measured center of pressure (CoP), anteroposterior (AP) and mediolateral (ML) postural sways during eight different balance tests (BALT’s). BALT's were designed to alter or cancel the systems involved in postural control: visual, vestibular and somatosensory. Subjects were randomly assigned to a caffeine group and an energy drink group. MANOVA analysis was performed for all variables of interest. Results: In the caffeine group, the AP sway of the Eyes Closed test on a stable surface was statistically significant. In the energy drinks group, we observed a general tendency of participants to increase CoP slightly, AP and ML sway in most of the BALT’s after the consumption of an energy drink. However, this increase was not statistically significant. These results suggest that in healthy young adults, the sensory re-weighting mechanism can overcome postural perturbation and maintain overall postural control. Conclusions: We observed an overall tendency to increase postural instability after the ingestion of energy drinks.", "keywords": [ "Caffeine", "Energy Drinks", "Postural Control", "Balance", "Re-Weighting mechanism", "CoP", "Sway" ], "content": "Introduction\n\nConsumption of energy drinks continues to increase since its appearance in the United States in 19971. Energy drinks are available in more than 140 countries; and by 2006, around 500 brands of energy drinks were established, where Thailand led the world in energy drinks consumption per person and the United States in the total volume of sales2,3. The main active ingredient in energy drinks is caffeine, a central nervous system stimulant (CNS) and the most widely used psychoactive drug in the world4. In addition to their main active ingredient caffeine, of which intake in high doses can alter the CNS, they also contain other substances such as3,4: taurine, guarana, cocoa, riboflavin, pyridoxine, nicotinamide, among other derivatives of herbs, which often contain additional amounts of caffeine4. For instance, guarana, in addition to its high caffeine content, contains theobromine and theophylline, which are mild CNS stimulants, that have been proven to cause higher levels of stimulation than caffeine alone in Dugesia tigrina, a free-living aquatic flatworm, with a CNS comparable to those found in mammals5.\n\nOne of the biggest problems with energy drinks is that manufacturers are not required by law to label the caffeine content of these additives. Sometimes they are just simply included in what they call an \"energy blend.\"4,6. It is not clear whether these additional ingredients provide a physical or cognitive improvement even more than the one provided by caffeine alone or how they affect the CNS4. How energy drinks affect the postural control and which sensory system (proprioceptive, visual, vestibular) is the most affected, remains unknown. Postural control is defined as the act of maintaining or restoring the achievement of postural control in any posture or activity through a complex interaction of the neurological and musculoskeletal system7,8. Because the central nervous system is responsible for maintaining postural control we believe that energy drinks, a CNS stimulant, could cause alterations in how the body reacts to different postural perturbations during stance. Therefore, the purpose of this study was to determine the energy drinks effects in the CNS postural control mechanism after sensory disturbances.\n\n\nMethods\n\nTrial registered at ClinicalTrials.gov: NCT03315442\n\nRegistration date: November 16, 2017\n\nA completed CONSORT checklist can be found in Supplementary File 1.\n\nThis study was approved by the Institutional Review Board of the UPR-MSC Human Research Protection Office (A2540116). Subjects of this study were recruited through advertisements published around the University of Puerto Rico, Medical Sciences Campus and Facebook page of the physical therapy students involved in this study. The study was carried out in the Biomechanics Laboratory of the Doctoral Program in Physical Therapy at the School of Health Professions in the Medical Sciences Campus, University of Puerto Rico (UPR-MSC).\n\nSubjects were invited to participate in the study through word of mouth, flyers and social media announcements during May 1, 2016 to December 15, 2016 for trial and recruitment purposes. Contact information was provided and potential participants called the Principal Investigator (PI) to express their interest in participating and schedule an appointment. Subjects were instructed to assist the biomechanics laboratory at the UPR-MSC. During the scheduled meeting, the PI explained the details of the study and when participants choose to partake there were given the informed consent. They had no specific time to finish reading informed consent, and they were encouraged to ask any questions during that process. When participants communicated to the PI that they finished reading the informed consent, the PI asked questions related to the study to ensured subjects did understand the purpose of the study and their role in the study.\n\nThe inclusion criteria for the study were designed to ensure a homogenous sample among the participants. 1) subjects between 21 to 40 years of age, 2) functional flexibility in the lower extremities, 3) functional muscular strength in the lower extremities, 4) completing the AHA/ASCM Health/Fitness Facility Pre-participation Screening Questionnaire, 5) a Body Mass Index of 18.5 to 29.9, 6) arterial blood pressure less than or equal to 140/90 mmHg, 7) pulse at rest between 45–90 beats per minute (BPM), and 8) 95% or more of oxygen saturation (spO2).\n\nThe exclusion criteria were designed to identify any factor that could alter postural control, other than energy drinks, or endanger the participant's safety. 1) subjects under the age of 21 and/or over the age of 40, 2) answering any questions affirmative on the pre-participation questionnaire, 3) cardiovascular problems, 4) severe balance problems, 5) taking any sedative or stimulant medications, 6) medical history of any neurological condition, 7) a fall in the last 3 months, 8) having undergone a surgical procedure in the past 6 months, 9) pain in the lower extremities and / or lower back, 10) have suffered a lower back injury and/or in the lower extremities in the last 6 months, 11) is pregnant or suspecting pregnancy, 12) have experienced adverse effects after caffeine consumption, 13) caffeine consumption 12 hours prior to the study intervention, 14) allergies to one of the energy drink ingredients, and 15) people unable to consent.\n\nAfter signing the informed consent and reviewing the inclusion and exclusion criteria, participants were subjected to a preliminary screening of participation using a list of questions and the AHA / ACSM Health / Fitness Facility Questionnaire9. This questionnaire's purpose is to ensure the safety of the participants to engage in physical activity by assessing the subjects and family history of cardiovascular diseases. Afterwards, we assessed subjects’ vital signs, blood pressure, pulse, and spO2, to ensure subjects were able to participate in physical activity safely ingest a moderate intake of caffeine without complications. Since postural control is negatively correlated with increased adiposity, weight and height data were measured to obtain a classification according to the Body Mass Index. A range of 18.5 to 29.9 was required to participate10,11. During the physical examinations the subjects also performed a Romberg test to rule out any obvious impairment in static balance, a modified Sit and Reach Test for the evaluation of functional flexibility, Sit to Stand Test (30 Seconds) for the assessment of lower limb functional strength, and a Tecumseh Step Test to evaluate the response of the cardiovascular system to a submaximal cardio test12–15. After the physical examination, the participants had a rest period between 10–20 minutes before starting the balance tests protocol of (BALT's).\n\nTwenty-three people contacted the researchers to participate in the study (see Supplementary File 2). We excluded 3 subjects from participating in the study for the following: 1) BMI of 34 (male), 2) did not pass pre-participation questionnaire for reporting adverse effects after ingesting caffeine (female) and 3) high blood pressure (male). Of the twenty participants, eleven (55%) were men and 9 (45%) women, with an average of 170.73 pounds, 26.1 years of age and 67.1 inches of height. The caffeine group consisted of 5 (50%) male participants and 5 (50%) female participants, while the energy drink group consisted of 6 (60%) female and 4 (40%) male participants (Figure 1).\n\nThe 20 healthy young subjects selected, were randomly assigned (simple randomization, flipping a coin 3 times, heads was energy drink, tales was caffeine) to control (caffeine) group (n = 10) and experimental (energy drinks) group (n = 10). Vital signs were retaken before the balance test protocol to ensure that the subjects remained within the previously established inclusion values.\n\nEach participant from both groups performed 8 balance tests (BALT's) which alter or cancel, individually or combined, sensory input from the sensory systems involved in postural control (Table 1). The order of the tests was changed systematically between subjects; therefore, they did not perform the tests in the same order to eliminate external factors such as fatigue or accommodation to the BALT's, which could alter the results. The BALT’s were conducted on the MatScan,™ (TekScan, Boston, MA) a pressure platform containing sensors that measure a displacement of the center of pressure in centimeters square (cm2) (CoP), anteroposterior (AP), and mediolateral (ML) sway in centimerters (cm)16. The data collected from the pressure mat was analyzed with Tekscan Sway Analysis Module (SAM) software designed for this purpose. The subjects stood for 30 seconds on the pressure platform during each test.\n\nAssessed= (+); Altered= (-). HUD*= Head up/down movements using a metronome 2/4, 60 BPM.\n\nThe first 4 BALT’s were carried out by placing the pressure platform on the floor, a stable surface. These tests were: 1) Open Eyes (EO) with a fixed point to evaluate all the systems involved in the postural control (visual, vestibular, somatosensory); 2) Eyes Closed (EC) to evaluate the vestibular and somatosensory system, while eliminating the visual sensory input; 3) Eyes Open while actively moving the head up and down (HUD) to evaluate the visual and somatosensory system, while altering the vestibular system with head movements (EO HUD) (For HUD movements a metronome 2/4 60 BPM was used to maintain a fixed frequency of about one spin per second in motion); 4) Eyes closed and actively moving the head up and down (EC HUD) to assess the effect of removing the visual input, in combination of an alteration of the vestibular system with the head up and down movements.\n\nThe remaining 4 BALT’s were the same as the 4 previously mentioned tests with the difference that the subject stood on an unstable surface (foam mat) that was placed on top of the pressure platform to alter the somatosensory (proprioceptive) system. The BALT’s on the unstable surface were: 1) Open Eyes (MAT EO), standing on the unstable surface to evaluate the visual and vestibular system while the somatosensory is altered; 2) Eyes Closed (MAT EO), standing on the unstable surface to evaluate the vestibular system (the somatosensory was modified and the visual system removed); 3) Eyes Open (MAT EO HUD) while actively moving the head up and down to evaluate the visual system, while altering the vestibular and somatosensory system; 4) Eyes Closed (MAT EC HUD) while actively moving the head up and down (in this test all three systems were altered). The same frequency of motion used for BALT’s 3 and 4 in the stable surface (1 spin per second, 60 BPM) was maintained for the BALT’s with HUD movements on the unstable surface.\n\nAfter the initial 8 pre BALT's, to the experimental group, 160 mg of caffeine was given through one energy drink (16 ounces). Monster energy drink was chosen because, unlike Redbull previously used by Enriquez, the label of Monster Energy drink exhibits the guarana additive, a potential CNS stimulant, in its nutritional label17,18. To the control group, we gave a caffeine pill of 200mg, a moderate dose in healthy adults which has been used in numerous studies on different topics and has not been associated with adverse effects such as toxicity, cardiovascular effects, behavior changes, among other things18. Because peak absorption of caffeine is reached within 30–45 minutes after the ingestion of caffeine/energy drink, there was a rest period of 30 minutes19,20. Vital signs were measured after the rest period, for the third time. The same 8 BALT’s were then performed, for post ingestion results.\n\nWe used the statistical package for the social sciences (SPSS) version 19 for all the data analysis. In this study, we used \"Teskcan Sway Analysis Module (SAM)\" software for our data collection. For data analysis, we first did a Shapiro-Wilks test to determine normality and to eliminate atypical values, or outliers for the CoP and sway data. Then, a paired Student’s T-test was used to compare values of weight, age, height and BMI of both groups and to determine homogeneity between the two groups. Concerning sway and CoP data, we used a MANOVA for all the variables of interest. A comparison Pre Caffeine/energy drink between groups (CoP and Sway) during all task was performed to ensure similarities between groups. Subsequently, a comparison between groups (MANOVA) was performed post Caffeine/energy drink consumption to assess the role of both substances. The test results were adjusted using a Bonferroni correction to identify the different parameters with a significant difference. In this study, a P value of 0.05 or lower was considered statistically significant.\n\n\nResults\n\nA Paired Student T-test (Table 2) was used to compare values of weight, age, height and BMI of both groups. None of the variables showed statistically significant results, evidencing homogeneity in the characteristics of subjects between the groups.\n\nIn the comparison of the caffeine group (Table 3), a significant increase (p ≤0.05) was found only in the AP sway in the eyes closed test while standing on a stable surface. The AP sways before caffeine ingestion was 1.65 ± 0.41, and 30 minutes after the ingestion it was 2.85 ± 1.1. For CoP in any of the BALT’s, including the EO test, the results were statistically significant.\n\nWhen comparing pre and post average of AP sway, ML sway and COP in the energy drinks group (Table 4), no statistically significant results were found in any of the variables measured in any of the BALT’s.\n\nNo statistically significant results were found for the comparison of the average of AP sway, ML sway, and COP of energy drinks and caffeine post BALT’s (Table 5).\n\nIn a secondary analysis, a comparison of the data over time (0, 15, 30 seconds) of each balance test was performed. In this analysis, we also observed a tendency of the energy drinks to alter the postural control, more significantly in the middle of the test and before subjects were able to recover overall postural control at the end of the test.\n\n\nDiscussion\n\nPostural control is a complex and dynamic interaction in which the CNS continuously receives afferent information from the vestibular, somatosensory, and visual systems. Once received by the CNS a relative weight is placed in these sensory inputs depending on the environment and goal of the task, resulting in a motor outcome required for postural control21. Different studies have found that weight or contribution of the 3 systems changes depending on the perturbation during stance22.\n\nThis shift in input and adjusting process, required to maintain control of the body, is referred as sensory re-weighting23. Several studies have demonstrated that sensory re-weighting is a major contributor to limiting body sway amplitudes when postural control is perturbed22. The purpose of this study was to assess the interaction of energy drinks on standing postural control. To determine the effects of energy drinks in the CNS postural control mechanism after sensory perturbations, we divided our analysis into several components.\n\nFirst, we assessed postural stability (pre and post average) and compared the effects of caffeine and energy drinks in the individual groups to determine the effects of consumption individually during eight sensory conditions.\n\nIn the caffeine pre and post comparison, 5/8 of the BALT's results showed an increase in the average of CoP. However, none of the results were statistically significant. A statistically significant difference (P≤0.05) was found only in AP sways values in the EC test in the stable surface. Nevertheless, the average of CoP in the same test did not show statistically significant difference suggesting that overall postural control was maintained. Even though in the present study the consumption of caffeine alone did not have a significant effect in altering the postural control, available evidence is inconsistent with this conclusion. In their study, Kim et al.24 measured postural control during EO, EC and a changed base of support after an approximate intake of 73 mg of caffeine. They concluded that after 40 minutes of caffeine intake, there was not a significant alteration to postural control in the healthy subjects. Compared with our study, they gave an amount of caffeine which was significantly less than the caffeine administrated in our study (200mg); but overall results were similar. Meanwhile, following the present study, Mcnerney et al.25 measured postural control after altering the system's associate to postural control after the ingestion of 300mg of caffeine and found a significant difference in the eyes closed, platform sway-referenced test. However, authors made a general conclusion that caffeine did not produce a clinically significant effect in healthy young participants. Furthermore, Franks et al.26 measured body sway of young subjects during stance with EO and EC after ingesting 300mg of caffeine per 70 kg of weight. The researchers observed a significant increase in body sway (eyes open) after 40 minutes, but subjects recovered stability at later times (100, 160 minutes). A similar behavior was observed with the eyes closed test of this study; but it was not significant, evidencing a tendency of the subjects to improve postural stability with time. It is important to note that in Franks et al. study, subjects’ weight was considerate to determine the amount of caffeine administrated, which should be taken into consideration for the comparison of the results of our study, and for the design of future studies assessing the effect of caffeine in postural control.\n\nIn the energy drinks pre and post comparison, 6/8 of the BALT's showed an increased average of COP, 5/8 BALT’s in the AP sway and 6/8 BALT’s in the ML sway 30 minutes after the consumption of the energy drink. The previous results suggest a negative effect on the postural control mechanism due to a possible stimulatory effect of the energy drinks in the CNS. The BALT’s EO, EC, EO HUD, EC HUD, EC MAT were affected in the three measurements (CoP, AP sway, ML sway), while EC HUD MAT was only affected in the CoP, and EO HUD MAT in the ML sway only. EO MAT was the only BALT’s not altered in any of the three measurements. Even though we observed a tendency of an increased postural instability after the energy drinks consumption, none of the results were statistically significant. No association between the systems altered, and the results could be established since there was not a pattern of the altered/canceled system and the increased instability observed in the BALT's. Enriquez et al.17 also measured body sway with the eyes closed and eyes open only 1 hour after the ingestion of energy drinks (160 mg of caffeine). In the study, 23 young subjects presented an increased sway in both conditions, but none of the results were statistically significant which correlates with our findings. Available evidence at the moment seems to indicate that energy drinks appear to make small increases in body sway. However, it does not impair the postural control mechanism significantly, at least in healthy subjects with a dose of 160 mg of caffeine given through energy drinks. How the postural control mechanism responds to an even higher dose of energy drinks or in other populations at risk for falls such as older adults remains unknown, however a stepping stone for future studies.\n\nSecondly, we compared the effects of caffeine versus energy drinks, to determine any differences in post test results regarding postural instability between the subjects of both groups, even though there was a difference of 40 mg of caffeine between the energy drink (160 mg) and caffeine group (200 mg). The average of CoP, AP sway and ML sway in the caffeine group seems to be more affected in the BALT's performed on the unstable surface (foam mat), while the energy drinks showed more instability in the stable surface. Albeit, an association or correlation between the system altered/canceled (specific BALT’s) and the effect between both groups could not be established, as the results were not consistent. Although, the energy drinks, in addition to the 160 mg of caffeine, contain potential stimulating ingredients. However, the difference of 40 mg of caffeine between groups was not a major factor as none of the post test results (CoP, AP sway, and ML sway) were statistically significant in the comparison between subjects of both groups. The results of this comparison show that the sensory re-weighting system of the twenty healthy young subjects in both groups was able to adjust and re-adjust the sensory inputs appropriately to maintain overall postural control despite the postural perturbations during the BALT’s and the stimulatory effect of the intake of the CNS stimulants caffeine and energy drinks.\n\n\nConclusion\n\nWe concluded that consumption of one 16 ounces energy drink does not impair the postural control mechanism significantly in healthy young adults. However, we observed an overall tendency to increase postural instability after the ingestion of one (16 ounces) energy drinks. We believe that our study adds to the working literature related to the effect of energy drinks and caffeine on posture balance and clarifies some of the inconsistencies related to this topic. Also, this study is a stepping stone for future studies related to motor control and the effects of external substance like energy drinks and caffeine. For instances, investigate postural control with a greater amount (ounces) of energy drink. Secondly, the habitual energy drink or caffeine consumption of the subjects prior the study was not measured; thus, we recommend adding this factor for future research as well. Thirdly, assess the effects on dynamic or gait balance control after caffeine or energy drink ingestion. Fourthly, future studies could consider taking balance measurements at earlier times, and more than once post consumption of energy drinks and caffeine. Fifth, we only included healthy young people. Additional studies can investigate the effects of the protocol of this study in other populations at fall risk: for instance elderly and subjects with sensory impairment or neurologic conditions. One final note, we did not assess the impact of caffeine or energy drinks in the different balance test using, as a baseline or control, the EO test compared to the other BALT's on a firm surface and the same for eyes on an unstable surface. We believed this information could tell us the impact of energy drink/caffeine on the different sensory systems.\n\n\nData availability\n\nDataset 1: Postural control dataset. doi, 10.5256/f1000research.12565.d18474427", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nThis article was published with support from Texas Woman's University Libraries’ Open Access Fund. Also, thanks to the volunteers who collaborated as participants in this study. Special thanks to the physical therapists who assisted and the MatScan™ equipment.\n\n\nSupplementary material\n\nSupplementary File 1: CONSORT Checklist.\n\nClick here to access the data.\n\nSupplementary File 2: CONSORT Flow Diagram.\n\nClick here to access the data.\n\n\nReferences\n\nMacdonald J: The potential adverse health effects of energy drinks. Am Fam Physician. 2013; 87(5): 321. PubMed Abstract\n\nIbrahim NK, Iftikhar R: Energy drinks: Getting wings but at what health cost? Pak J Med Sci. 2014; 30(6): 1415–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nReissig CJ, Strain EC, Griffiths RR: Caffeinated energy drinks--a growing problem. Drug Alcohol Depend. 2009; 99(1–3): 1–10. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSeifert SM, Schaechter JL, Hershorin ER, et al.: Health effects of energy drinks on children, adolescents, and young adults. Pediatrics. 2011; 127(3): 511–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoustakas D, Mezzio M, Rodriguez BR, et al.: Guarana provides additional stimulation over caffeine alone in the planarian model. PLoS One. 2015; 10(4): e0123310. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nChilds E: Influence of energy drink ingredients on mood and cognitive performance. Nutr Rev. 2014; 72 Suppl 1: 48–59. PubMed Abstract \| Publisher Full Text\n\nShumway-Cook A, Woollacott MH: Motor Control: Translating Research Into Clinical Practice. Lippincott Williams & Wilkins; 2012. Reference Source\n\nPollock AS, Durward BR, Rowe PJ, et al.: What is balance? Clin Rehabil. 2000; 14(4): 402–6. PubMed Abstract \| Publisher Full Text\n\nBalady GJ, Chaitman B, Driscoll D, et al.: Recommendations for cardiovascular screening, staffing, and emergency policies at health/fitness facilities. Circulation. 1998; 97(22): 2283–93. PubMed Abstract \| Publisher Full Text\n\nKu PX, Abu Osman NA, Yusof A, et al.: Biomechanical evaluation of the relationship between postural control and body mass index. J Biomech. 2012; 45(9): 1638–42. PubMed Abstract \| Publisher Full Text\n\nSingh D, Park W, Levy MS, et al.: The effects of obesity and standing time on postural sway during prolonged quiet standing. Ergonomics. 2009; 52(8): 977–86. PubMed Abstract \| Publisher Full Text\n\nRaad J: Rehab Measures: Romberg Test. Rehabilitation Measures Database. Published: January 23, 2014. Actualized: August 28, 2014. Accessed: May 13, 2016. Reference Source\n\nRaad J: Rehab Measures: 30 Seconds Sit to Stand Test. Rehabilitation Measures Database. Published: December 18, 2013. Actualized: January 31, 2014. Accessed: May 15, 2016. Reference Source\n\nMackenzie B: Tecumseh Step Test. BrianMac Sports Coach. Accessed: May 10, 2016. Reference Source\n\nMackenzie B: Sit and Reach Test. BrianMac Sports Coach. Accessed: May 10, 2016. Reference Source\n\nBrenton-rule A, Mattock J, Carroll M, et al.: Reliability of the TekScan MatScan® system for the measurement of postural stability in older people with rheumatoid arthritis. J Foot Ankle Res. 2012; 5: 21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEnriquez A, Sklaar J, Viirre E, et al.: Effects of caffeine on postural stability. Int Tinnitus J. 2009; 15(2): 161–3. PubMed Abstract\n\nMcLellan TM, Lieberman HR: Do energy drinks contain active components other than caffeine? Nutr Rev. 2012; 70(12): 730–44. PubMed Abstract \| Publisher Full Text\n\nBispo MS, Veloso MC, Pinheiro HL, et al.: Simultaneous determination of caffeine, theobromine, and theophylline by high-performance liquid chromatography. J Chromatogr Sci. 2002; 40(1): 45–48. PubMed Abstract \| Publisher Full Text\n\nMiners JO, McKinnon RA: CYP1A. In: Levy RH, Thummel KE, Trager WF, Hansten PD, Eichelbaum ME, editors. Metabolic Drug Interactions. New York, NY USA: Lippincott Williams & Wilkins; 2000; 61–73.\n\nHorak FB: Postural orientation and equilibrium: what do we need to know about neural control of balance to prevent falls? Age Ageing. 2006; 35 Suppl 2: ii7–ii11. PubMed Abstract \| Publisher Full Text\n\nAssländer L, Peterka RJ: Sensory reweighting dynamics in human postural control. J Neurophysiol. 2014; 111(9): 1852–64. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPradels A, Pradon D, Hlavačková P, et al.: Sensory Re-Weighting in Human Bipedal Postural Control: The Effects of Experimentally-Induced Plantar Pain. PLoS One. 2013; 8(6): e65510. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim WS, Choi CK, Yoon SH, et al.: Usual Dose of Caffeine Has a Positive Effect on Somatosensory Related Postural Stability in Hemiparetic Stroke Patients. Ann Rehabil Med. 2014; 38(6): 775–783. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMcNerney KM, Coad ML, Burkard RF: The influence of caffeine on the sensory organization test. J Am Acad Audiol. 2014; 25(6): 521–528. PubMed Abstract \| Publisher Full Text\n\nFranks HM, Hagedorn H, Hensley VR, et al.: The effect of caffeine on human performance, alone and in combination with ethanol. Psychopharmacologia. 1975; 45(2): 177–181. PubMed Abstract \| Publisher Full Text\n\nRosario MG, Collazo H, Mateo M, et al.: Dataset 1 in: Increased static postural sway after energy drink consumption: A randomized trial. F1000Research. 2017. Data Source" }	[ { "id": "28251", "date": "08 Dec 2017", "name": "Ann Hallemans", "expertise": [ "Reviewer Expertise biomechanics", "movement science", "balance control", "gait" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors performed a study on the effect of caffeine intake on postural sway. They compared two situations: intake of caffeine by a pil (200 mg) and intake of caffeine (160 mg) by an energy drink containing additional ingredients with caffeine of which the amount of additional caffeine and modulating effects are unknown. They want to investigate whether the \"energy blend\" in energy drinks has a different effect on postural sway in different sensory modalities, compared to intake of pure caffeine. Given the frequent consumption of energy drinks, this seems like a valid question. What I would have expected, though, is a stronger motivation for why exactly postural control/ postural sway should be investigated. Do you expect an increased risk of falling or is it related to safety issues?\nThe study design is appropriate, although the sample size is relatively small. Subjects are their own controls, which makes the study stronger. It should be motivated, however, why the dose of caffeine in the energy drink (160 mg) is lower than the dose in the pil (200 mg). Why was the pil not dosed at 160 mg as well?\nRegarding the methodology, some additional details should be provided. Resolution and measurement frequency of the MatScan should be provided. Furthermore, did you analyse the entire 30s or leave out the first 10 s to eliminate start-up effects? Also provide size and density of the foam pad. You report that COP and sway data are outcome parameters. This is not specific enough. The reader would require to know which outcome parameters (amplitude, range, path, speed, area, ...) were used. Furthermore, information should be provided on preprocessing of the COP trajectory, e.g. filtering (which filter type, cut-off frequency, ...) as well as the formulas used to calculate the outcome parameters. Units of measurement should definitely be included when reporting outcome measurements.\nStatistical analysis is only partly appropriate. It is not clear whether the quality of data was checked and whether outliers were removed. The database contains some strange values. Furthermore, it is not correct to perform a paired t-test to compare two groups. This would require an independent samples t-test. The main statistical analysis was a MANOVA, which seems appropriate to answer the research question. Please define the model: which variables were dependents? which were factors? What main effects and possible interaction effects were investigated?\nI do not feel conclusions are very well supported by the data, which partially might relate to uncertainty about what parameters are actually investigated. In the tables it is unclear whether reported p-values are subject to Bonferonni correction or not. Furthermore, in the discussion results are reported as an increase in postural sway (p. 7/10) although results are not significant. If the difference is not significant, you cannot report it as an increase. The conclusion is reported correctly in that the data are not able to indicate a significant increase in postural sway after the consumption of caffeine. However, the title of the manuscript makes a very strong statement that suggests otherwise and this creates confusion.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? No", "responses": [] }, { "id": "29888", "date": "25 Jan 2018", "name": "Evelyn B. Voura", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nIn this interesting article Martin G. Rosario et al. examined the effect of caffeine on postural control via a pressure mat when a ‘system’ involved in this physiological reflex was compromised. The findings led the investigators to conclude that caffeine caused the study participants – all young healthy adults – to be less stable following caffeine injection. The motivation behind the study was the ever-increasing consumption of caffeinated energy drinks, leading researchers to help build an increased understanding of how these beverages might affect physiology – particularly since caffeine content is typically not fully disclosed on the packaging.\n\nStudy-Related Considerations:\n\nThe tests used by the investigators to assess postural control seem rigorous, and carefully considered. However, this reviewer is not familiar with the specifics of the data collected by these pressure platforms. To help readers fully appreciate these details, it might be beneficial to elaborate on the measurements taken by the device.\n\nThe primary drawback of such studies is often the sample size, and this is the case here as well. It would be ideal to canvas more participants, which of course would require more time for the study, given that the search was limited to the investigating campus. With the small sample size, however, meaningful data can be collected – the statistical analysis of choice should be the Student’s T-test for the before and after treatment comparison. The fact that the study participants were more similar than they were different, despite their gender as evidenced by the analysis of the homogeneity of the samples, lends more credence to the use of the Student’s T-test as a method of analysis – a Bonferroni correction is helpful, as was reported by the authors. Using the T-Test, however, to assess how similar the participants were, seems out of place – this reads like it would be more appropriate for the analysis of variance. So to this reviewer, the statistical choices were reversed. Comparing the participants as their own controls (before and after) instead of a pool of ‘treated and untreated’ unrelated individuals is helpful. However, the drawback is the low level of significance reported in Table 3. To really understand the effect of the caffeine in these measurements, a clearer level of significance is needed. It would be of interest, therefore, to determine the findings with the other statistical test. A larger sample size, of course, can also help, but that might not be possible for the investigators to consider at this time. In the end, it may be that energy drinks and caffeine at these concentrations does not have any effect on postural control as the other publications referenced by the authors note. However, given that these studies are still underway by this and other groups, despite other corroborating evidence, there does seem to be some question into these conclusions. Indeed, despite statistics, which show the contrary in almost all cases, the authors still believe that their study subjects were less stable following the supplementation. As such, it is worth ensuring that the statistics are rigorous to settle the discussion.\n\nThe authors might also consider presenting their data – the significance levels - in a histogram format – so that the reader can more quickly identify those tests providing the greatest effect – and also directly compare caffeine versus energy drink. This is particularly helpful when the significance values themselves are not considerable.\n\nThe other point worth noting is the difference in the level of caffeine used for the energy drink group and the caffeine pill group. It is understandable that the authors desired to have their findings be comparable to previous studies using the 200 mg level, but it is also difficult to compare the effect of 160 mg with 200 mg. This reviewer also wonders if the form of the solution – water or an energy drink formulation – pill matrix versus soluble, might also affect the absorption of caffeine. Was the fact that the only significant effect measured by the investigators in Table 3 due to the caffeine level, or due to the formulation of a pill versus an energy drink? Using the same concentration might help to eliminate this confounding factor. The authors do make note of other studies that use weight at a determining factor toward how much caffeine should be administered, however, since the idea is to study the effect of energy drinks (which are consumed by individuals regardless of weight), and because the control for each individual is the subject’s own reactions without the supplementation, this reviewer does not find the method of dosing the subjects of particular concern.\n\nAnother idea worth considering is the time to effect. The authors mention this interest as well in their discussion. It would be interesting for this reviewer, for example, to see what the effect would be if the participants were also tested one hour or so afterward as well. Is there an effect due to the ‘let down’ of the caffeine that could be detected for example – a ‘crash’ effect? The investigators observed, in their secondary analysis, mentioned prior to the final Dataset 1 link, that there were detectable differences in postural control between the caffeine and energy drink groups during the test, and that the subjects recovered control by 30-second end time. This implies a difference between these two groups that is speculated upon, but not documented – and perhaps would lend more information to the entire story of how caffeine and energy drinks affect physiology. Since the authors have the data, why not report the results at different intervals during the test and not only from those numbers collected at the end? This might provide a better picture of the effect of a) caffeine in general, and b) the energy drink formulation where more than just the caffeine may be at play. Even if the findings from different times prove to also not be statistically significant by the standards given, the trends in the data with time, if shown graphically, might be particularly informative for future studies - and aid the reader with a means to gain a complete ‘picture’ of what the authors observed while running their study, and why they arrived at their conclusions, despite their statistical analysis.\n\nGrammatical Considerations:\n\nThere are a few textual items to be corrected in an updated version of the work. While cosmetic, these minor corrections will improve the overall quality of the document. By way of example: Under Ethical statement: 1) “…choose to partake there were given…” should read “…chose to partake they were given…” 2) “…asked questions related to the study to ensured subjects did understand…” should read “asked questions related to the study to ensure subjects understood…”. The authors should have a careful read of the text to ensure these or other grammatical issues do not distract the reader from their findings.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [ { "c_id": "3920", "date": "29 Aug 2018", "name": "Martin Rosario", "role": "Author Response", "response": "Dear Dr. Voura,Thank you for your feedback. To answer the question regarding the difference in the caffeine mg, this idea is because we wanted to compare the effects of the equivalent to a cup of coffee (200 mg caffeine pill) versus an energy drink (standard of 160 mg) and how these affected postural sway. Also, the study was designed to be an initial pilot study and therefore the number of subjects is small. However, we do plan on continuing the study and to increase the amount of subjects.Regarding table 3, we were also as surprised to see that the levels of significance were not quite what we were expecting. This will be addressed in our future studies by increasing the amount of subjects, reporting the time-frame/time-effect approach, and observing other variables. However, it gives us an indication of what route to take in the future. Also, the suggestion of a histogram is a great idea that we may consider for our future studies. Finally, thank you for noticing the grammatical errors. We do use other peers and electronic tools to revise the grammar, but we missed that. Thank you.Dr. Gonzalez" } ] } ]	1	https://f1000research.com/articles/6-2036
https://f1000research.com/articles/5-1919/v1	05 Aug 16	{ "type": "Research Note", "title": "Predicted protein interactions of IFITMs which inhibit Zika virus infection", "authors": [ "Madhavi K. Ganapathiraju" ], "abstract": "After the first reported case of Zika virus in Brazil, in 2015, a significant increase in the reported cases of microcephaly was observed. Microcephaly is a neurological condition in which the infant’s head is significantly smaller with complications in brain development. Recently, two small membrane-associated interferon-inducible transmembrane proteins (IFITM1 and IFITM3) have been shown to repress members of the flaviviridae family which includes the Zika virus. However, the exact mechanisms leading to the inhibition of the virus are yet unknown. Here, we assembled an interactome of IFITM1 and IFITM3 with known protein-protein interactions (PPIs) collected from publicly available databases and novel PPIs predicted using High-confidence Protein-Protein Interaction Prediction (HiPPIP) model. We analyzed the functional and pathway associations of the interacting proteins, and found that there are several immunity pathways (interferon signaling, cd28 signaling in T-helper cells crosstalk between dendritic cells and natural killer cells), neuronal pathways (axonal guidance signaling, neural tube closure and actin cytoskeleton signaling) and developmental pathways that are associated with these interactors. These results could help direct future research in elucidating the mechanisms underlying the viral immunity to Zika virus and other flaviviruses.", "keywords": [ "Zika", "virus infection", "protein interaction", "interferon-inducible transmembrane proteins" ], "content": "Introduction\n\nThe Zika virus (ZIKV) is a flavivirus that was initially isolated from rhesus monkeys in 1947 and was first reported in humans in 19521. Until recently, reports of this virus had been limited to Africa and Asia2 but currently there is an ongoing, wide-spread Zika epidemic3. The virus has rapidly spread across the Americas and has been declared a ‘global emergency’ by the World Health Organization4. It is mostly transmitted by mosquitoes and clinical manifestations include rash, mild fever, arthralgia, conjunctivitis, myalgia, and headaches. In addition, it has been reported recently that the virus can be transmitted sexually, with the risk of infection persisting for several months after initial contact5. Earlier, the symptoms of ZIKV had been reported to be mild1, but, the virus has been recently linked to two more serious afflictions: Guillen-Barré syndrome6,7 and microcephaly8–12, both of which are serious neurological conditions. Microcephaly results in reduced head circumference measurement in infants, exhibiting complications in brain development. Of particular concern is the attribution of microcephaly to infection with ZIKV occurring between the first two trimesters of pregnancy11,12. Evidence linking ZIKV to microcephaly includes detection of ZIKV RNA in tissue such as the placenta and amniotic fluid of pregnant women with ZIKV, as well as in the brains of stillborn infants with microcephaly13. In a study with human induced pluripotency stem cells, the mechanism of ZIKV related cell death has been elucidated. This study demonstrated that ZIKV infects human embryonic cortical neural progenitor cells (hNPCs), ultimately leading to attenuated population growth mediated by virally induced caspase-3-mediated apoptosis and cell-cycle dysregulation14. Mice studies showed that ZIKV infection can lead to nerve degeneration, softening of the brain and porencephaly15.\n\nVery recently, two small membrane-associated interferon-inducible transmembrane proteins (IFITMs) IFITM1 and IFITM3 were discovered to have a protective role against the Zika virus infection by inhibiting replication of the virus and preventing cell death induced by Zika virus5. IFITMs were shown to have an inhibitory role against other flaviviruses also, such as West Nile and dengue virus. Type 1 interferon (IFN) signaling inhibits Zika virus pathogenesis. Prior to induction of IFN-stimulated genes, IFITMs may provide initial defense against the infection5. However, since the exact mechanism of IFITM1 and IFITM3 mediated restriction are yet unknown, computational methods could accelerate research by presenting testable hypotheses.\n\nIn our earlier work, we developed a computational model called ‘High-confidence Protein-Protein Interaction Prediction’ (HiPPIP) model that identifies novel protein-protein interactions (PPIs) in the human interactome16, motivated by the fact that PPIs prove to be valuable in understanding the function of a gene, and specifically in how it plays a role in causing or preventing disease. One example of the impact of these computational predictions is the PPI that we predicted between OASL and RIG-I16, which was validated to be a true PPI through co-immunoprecipitation16,17. This led to the formulation of a hypothesis about its significance and led to the discovery of its functional relevance, namely that upon viral infection, OASL triggers the immune system by activating the RIG-I pathway, thus inhibiting virus replication18. functional studies initiated solely by this predicted PPI showed that human OASL binds to dsRNA to enhance RIG-I signaling, and that boosting OASL can help inhibit viral infection18. In this work, we applied HiPPIP model to discover novel PPIs of IFITM1 and IFITM3, to potentially accelerate the discovery of the mechanism by which they inhibit ZIKV and other viral infections.\n\n\nMethods\n\nPPIs were assembled by collecting known PPIs from the Human Protein Reference Database (HPRD)19 and Biological General Repository for Interaction Datasets (BioGRID)20, and by computing novel PPIs using the HiPPIP model that we developed16. Computationally discovered PPIs have been shown to be highly accurate by computational evaluations and experimental validations of a few PPIs16. Interactome figures were created using Cytoscape21. Pathways associated with proteins in the interactome were collected using Ingenuity Pathway Analysis® suite (http://www.ingenuity.com). Gene Ontology terms enriched among the interacting partners (including the candidate genes IFITM1 and IFITM3) were computed using the BiNGO plugin of Cytoscape22.\n\n\nResults and discussion\n\nWe assembled the PPIs of IFITM1 and IFITM3 (Figure 1) by computing novel PPIs using HiPPIP model and collecting known PPIs from publicly available databases, Human Protein Reference Database (HPRD) and Biological General Repository for Interaction Dataset (BioGRID)23,24. We found that both proteins have known PPIs with proteins involved in immunity, and several novel (predicted) PPIs with proteins that seem to have relevant functions. DEAF1 is involved in neural tube closure, embryonic skeletal development and anatomic structure morphogenesis, and other functions. FNDC3B was found to be associated with heart rate, height and corneal structure through genome-wide association studies. It is a membrane protein, and, while its own functions are unknown, its interactors are involved in regulation of glial cell apoptotic process, regulation of ion transport (sodium, potassium, calcium) and several cardiac processes. SPTA1 is involved in neural functions of actin filament organization, neurite outgrowth and axon guidance. RASSF7 is localized to microtubule organizing center. While its function is unknown, it interacts with proteins that are involved in cell proliferation in brain, regulation of neuroblast proliferation, nervous system development, synaptic vesicle fusion to presynaptic membrane, and viral budding and assembly. TSSC4 interacts with both IFITM1 and IFITM3. TSSC4’s functions are unknown but its own interactions suggest that it may be involved in viral penetration into host nucleus, protein import into nucleus and immune response signaling, among other processes. TLR7 is involved in several functions and pathways related to innate immunity. ARPC1B is part of actin related protein 2/3 complex; its interactions suggest that it may be involved in neuronal development such as axonogensis and development, neuron differentiation, nervous system development, and immune related terms such as innate immune response, regulation of immune response, etc. These functional annotations are sourced from Schizo-Pi16,25; for example, see: http://severus.dbmi.pitt.edu/schizo-pi/index.php/gene/view/10522.\n\nNovel interactors of IFITM1 and IFITM3 are shown as red colored nodes while previously known interactors are shown as light blue colored nodes. Novel interactors of IFITM1 and IFITM3 are shown as red colored nodes while previously known interactors are shown as light blue colored nodes.\n\nPathways associated with IFITM interactome computed with Ingenuity Pathway Analysis Suite® are given in Table 1. Gene Ontology biological process terms associated with the interactome, compiled with BiNGO22 are shown in Figure 2 and Table 2.\n\nPathway associations were computed with Ingenuity Pathway Analysis Suite ®. Novel interactors are shown in bold.\n\nYellow color signifies statistically significant enrichments. Novel interactors that are associated with the GO terms are shown in red and known interactors in blue. See Table 1 for a complete list of terms associated with the genes.\n\nNovel interactors are shown in bold.\n\nThere is only one study that presents altered gene expression under ZIKV infection available in Gene Expression Omnibus14. The study with eight samples (four infected and four control samples) showed that the infection of human neural progenitor cells (hNPCs) with the virus caused increased cell death and cell-cycle dysregulation14. We examined whether any of the interacting genes were differentially expressed in that study and found five genes that were differentially expressed with a small fold-change but with significant p-value (< 0.005) (Table 2): CD81, NME5, and RASSF7 were found to be under-expressed and FNDC3B and UIMC1 were found to be over-expressed (Table 3).\n\n\nOther resources\n\nSee http://severus.dbmi.pitt.edu/schizo-pi for annotations of individual proteins that are compiled from various databases. Also see the following link to our LENS webserver, where we present annotations of all the genes in the IFITM1-IFITM3 interactome and also annotations of proteins that further interact with interactors (i.e. 2nd level connectors of IFITMs). Under each tab, ‘candidate genes’ refers to IFITMs and their interactors shown in Figure 1 of the paper, while entire interactome includes all of their interactors. Note that the database behind LENS does not include novel protein-protein interactions; therefore, they are not shown as edges in the network diagram. The sources of the pathways and disease associations shown on this website are given in 25,26.\n\nhttp://severus.dbmi.pitt.edu/LENS/index.php/results/view/57649c2516f9a/admin_57649c251737d\n\n\nData availability\n\nAll pertaining data are provided in the manuscript.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work is funded by Biobehavioral Research Awards for Innovative New Scientists (BRAINS) grant (R01MH094564) from National Institute of Mental Health of National Institutes of Health of USA.\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nMKG thanks her students Josefina Correa Menendez, Mara Staines, Varsha Embar and Srilakshmi Chaparala for assisting in this work.\n\n\nReferences\n\nDick GW, Kitchen SF, Haddow AJ: Zika virus. I. Isolations and serological specificity. Trans R Soc Trop Med Hyg. 1952; 46(5): 509–20. PubMed Abstract \| Publisher Full Text\n\nHiggs S: Zika Virus: Emergence and Emergency. Vector Borne Zoonotic Dis. 2016; 16(2): 75–6. PubMed Abstract \| Publisher Full Text\n\nLazear HM, Diamond MS: Zika Virus: New Clinical Syndromes and Its Emergence in the Western Hemisphere. J Virol. 2016; 90(10): 4864–4875. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPetersen E, Wilson ME, Touch S, et al.: Rapid Spread of Zika Virus in The Americas--Implications for Public Health Preparedness for Mass Gatherings at the 2016 Brazil Olympic Games. Int J Infect Dis. 2016; 44: 11–5. PubMed Abstract \| Publisher Full Text\n\nSavidis G, Perreira JM, Portmann JM, et al.: The IFITMs Inhibit Zika Virus Replication. Cell Rep. 2016; 15(11): 2323–30. PubMed Abstract \| Publisher Full Text\n\nAraujo LM, Ferreira ML, Nascimento OJ: Guillain-Barré syndrome associated with the Zika virus outbreak in Brazil. Arq Neuropsiquiatr. 2016; 74(3): 253–5. PubMed Abstract \| Publisher Full Text\n\nCao-Lormeau VM, Blake A, Mons S, et al.: Guillain-Barré Syndrome outbreak associated with Zika virus infection in French Polynesia: a case-control study. Lancet. 2016; 387(10027): 1531–9. PubMed Abstract \| Publisher Full Text\n\nBroutet N, Krauer F, Riesen M, et al.: Zika Virus as a Cause of Neurologic Disorders. N Engl J Med. 2016; 374(16): 1506–1509. PubMed Abstract \| Publisher Full Text\n\nde Paula Freitas B, de Oliveira Dias JR, Prazeres J, et al.: Ocular Findings in Infants With Microcephaly Associated With Presumed Zika Virus Congenital Infection in Salvador, Brazil. JAMA Ophthalmol. 2016; 134(5): 529–535. PubMed Abstract \| Publisher Full Text\n\nHazin AN, Poretti A, Turchi Martelli CM, et al.: Computed Tomographic Findings in Microcephaly Associated with Zika Virus. N Engl J Med. 2016; 374(22): 2193–5. PubMed Abstract \| Publisher Full Text\n\nMlakar J, Korva M, Tul N, et al.: Zika Virus Associated with Microcephaly. N Engl J Med. 2016; 374(10): 951–8. PubMed Abstract \| Publisher Full Text\n\nMoron AF, Cavalheiro S, Milani H, et al.: Microcephaly associated with maternal Zika virus infection. BJOG. 2016; 123(8): 1265–1269. PubMed Abstract \| Publisher Full Text\n\nCarod-Artal FJ: Epidemiology and neurological complications of infection by the Zika virus: a new emerging neurotropic virus. Rev Neurol. 2016; 62(7): 317–328. PubMed Abstract\n\nTang H, Hammack C, Ogden SC, et al.: Zika Virus Infects Human Cortical Neural Progenitors and Attenuates Their Growth. Cell Stem Cell. 2016; 18(5): 587–590. PubMed Abstract \| Publisher Full Text\n\nWaddell LA, Greig JD: Scoping Review of the Zika Virus Literature. PLoS One. 2016; 11(5): e0156376. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanapathiraju MK, Thahir M, Handen A, et al.: Schizophrenia interactome with 504 novel protein-protein interactions. NPJ Schizophr. 2016; 2: 16012. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGanapathiraju M, Sweet RA, Mohamed TP: 166. Newly discovered protein–protein interactions of schizophrenia associated genes and their biomedical significance. Brain Behavior and Immunity. 2011; 25(Supplement 2): S227. Publisher Full Text\n\nZhu J, Zhang Y, Ghosh A, et al.: Antiviral activity of human OASL protein is mediated by enhancing signaling of the RIG-I RNA sensor. Immunity. 2014; 40(6): 936–48. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPeri S, Navarro JD, Kristiansen TZ, et al.: Human protein reference database as a discovery resource for proteomics. Nucleic Acids Res. 2004; 32(Database issue): D497–501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nStark C, Breitkreutz BJ, Reguly T, et al.: BioGRID: a general repository for interaction datasets. Nucleic Acids Res. 2006; 34(Database issue): D535–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShannon P, Markiel A, Ozier O, et al.: Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13(11): 2498–504. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMaere S, Heymans K, Kuiper M: BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 2005; 21(16): 3448–9. PubMed Abstract \| Publisher Full Text\n\nBreitkreutz BJ, Stark C, Reguly T, et al.: The BioGRID Interaction Database: 2008 update. Nucleic Acids Res. 2008; 36(Database issue): D637–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKeshava Prasad TS, Goel R, Kandasamy K, et al.: Human Protein Reference Database--2009 update. Nucleic Acids Res. 2009; 37(Database issue): D767–72. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOrii N, Ganapathiraju MK: Wiki-pi: a web-server of annotated human protein-protein interactions to aid in discovery of protein function. PLoS One. 2012; 7(11): e49029. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHanden A, Ganapathiraju MK: LENS: web-based lens for enrichment and network studies of human proteins. BMC Med Genomics. 2015; 8(Suppl 4): S2. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "15557", "date": "08 Aug 2016", "name": "Judith Klein-Seetharaman", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe title, abstract and article overall are well written and clear. The design, methods and analysis are mostly well described, although some detail could be added. In particular, given that the IFITM interactome contains a large number of previously unknown PPI’s, it would be useful to give a little more detail on the methodology on how these predictions were obtained, rather than just stating that HiPPIP was used. In particular, it would be useful to understand what cut-off was used and a brief mentioning of the prediction methodology in general. An estimate of false positive and false negative errors for the prediction in Figure 1 would be particularly helpful.\n\nIn the analysis, the legend for Figure 2 needs expansion. It is not clear what the edges signify and in particular what is the meaning of directionality in the arrows.\n\nI object to the wording used on page 2 “which was validated to be a true PPI”, as any PPI evidence is debatable. I would reword to “which was experimentally validated”.\n\nI also object to the wording used on page 2 “Computationally discovered PPIs have been shown to be highly accurate”. Such a blanket statement is clearly not true, as there are many predictions out there that are highly inaccurate. A more specific example needs to be provided here.\n\nA minor suggestion is to replace “HiPPIP model” with “the HiPPIP model”.", "responses": [] }, { "id": "17909", "date": "08 Dec 2016", "name": "Nicholas Eyre", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript reports a number of novel protein interactions of IFITM1 and IFITM3 proteins, as determined using a 'High-confidence Protein-Protein Interaction Prediction (HiPPIP)' computational prediction model of protein-protein interactions. Analysis of functional and pathway associations of the putative interacting proteins is also reported, as they may relate to IFITM-mediated restriction of Zika virus infection. The manuscript is generally well-written and the title is appropriate. However, there are several ways in which the study and manuscript could be improved:\nThe report is largely based upon the predictions generated by the HiPPIP model. As this model is not well-described here and has not been extensively validated experimentally in the literature, it would be helpful if the criteria/rules employed by the model were described (Are predictions based purely on structural features? Does the model take into account protein localization, regulation, tissue distribution, known interactions etc.?). In the Methods section it is simply stated that 'Computationally discovered PPIs have been shown to be highly accurate by computational evaluations and experimental validations of a few PPIs'. This statement is not well-justified and may be misleading. In support of the HiPPIP model, the author refers to the successful prediction of OASL interaction with RIG-I ('Functional studies initiated solely by this predicted PPI showed...'). However in the cited publication (on which Dr. Ganapathiraju is an author)1 the HiPPIP model (and associated publications) was not referred to. The HiPPIP model should be more clearly described here, including some analysis/discussion of its success rate in predicting protein-protein interactions that have been experimentally validated.\n\nAs this report focuses on IFITM1 and IFITM3 antiviral functions and predicted interactions, it would be useful if the features and properties of these proteins were at least briefly described (domains, membrane topology, post-translational modifications, sub-cellular localization, tissue distribution and regulation). This would help to interpret the significance of the predicted interactions.\n\nIn the Results and Discussion some of the roles/properties of the predicted interacting partners are listed. It would be helpful if references were provided for these functions/properties (even if only to reviews). Furthermore, it would be helpful if these properties were discussed in the context of Zika virus and/or IFITM biology (e.g. commonalities in tissue/sub-cellular distribution and/or regulation and features of these proteins [e.g. domains] that may support their predicted interactions).\n\nThe manuscript should be carefully edited to clarify interactions that are purely predicted and not supported by experimental evidence (e.g. statements like 'TSSC4 interacts with IFITM1 and IFITM3' are misleading).\n\nA conclusion/summary paragraph that briefly summarizes the major findings and their significance and potential future directions may be appropriate.", "responses": [] }, { "id": "17282", "date": "12 Jan 2017", "name": "Sandeep Chakraborty", "expertise": [], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript presents a use model of the a protein-protein interaction method developed by the author along with other collaborators, focused on a very important pathogen (Zika) in the present circumstances. It is lucidly written and well presented.\nThe biggest shortcoming of this manuscript is the failure to cite several previous work related to the interferon-inducible transmembrane protein 3 (\"The Antiviral Effector IFITM3 Disrupts Intracellular Cholesterol Homeostasis to Block Viral Entry\" - Amini-Bavil-Olyaee, et. al, 2013, \"The antiviral effector IFITM3 disrupts intracellular cholesterol homeostasis to block viral entry\", Amini-Bavil-Olyaee, et. al, 2013 to name a couple).\nFurthermore, the current manuscript and its stated methodology does not find two proteins (VAPA and OSBP) that have been shown to have interactions with IFITM3. This would have been a clincher. It would also be proper to mention other work related to IFITM mechanism (\"IFITM Proteins Restrict Viral Membrane Hemifusion\": Li et. al, 2013) - and focus on the viral membrane hemifusion mentioned there.\n\nMinor comments:\n\"functional studies initiated solely\" - capitalize functional.\n\nWidespread use of \"we\" with only a single author.\n\n\"Computationally discovered PPIs have been shown to be highly accurate by computational evaluations and experimental validations of a few PPIs'.\" - overstated and speculative.\n\nIt is also speculative to correlate the expression changes of predicted interactors to these IFITMs, since there can be multiple other reasons for such expression changes (\"whether any of the interacting genes were differentially expressed in that study and found five genes that were differentially expressed\").", "responses": [] } ]	1	https://f1000research.com/articles/5-1919
https://f1000research.com/articles/6-2032/v1	20 Nov 17	{ "type": "Research Article", "title": "A study on the efficacy of APACHE-IV for predicting mortality and length of stay in an intensive care unit in Iran", "authors": [ "Mohammad Ghorbani", "Haleh Ghaem", "Abbas Rezaianzadeh", "Zahra Shayan", "Farid Zand", "Reza Nikandish", "Mohammad Ghorbani", "Abbas Rezaianzadeh", "Zahra Shayan", "Farid Zand", "Reza Nikandish" ], "abstract": "Background: Clinical assessment of disease severity is an important part of medical practice for prediction of mortality and morbidity in Intensive Care Unit (ICU). A disease severity scoring system can be used as guidance for clinicians for objective assessment of disease outcomes and estimation of the chance of recovery. This study aimed to evaluate the hypothesis that the mortality and length of stay in emergency ICUs predicted by APACHE-IV is different to the real rates of mortality and length of stay observed in our emergency ICU in Iran. Methods: This was a retrospective cohort study conducted on the data of 839 consecutive patients admitted to the emergency ICU of Nemazi Hospital, Shiraz, Iran, during 2012-2015. The relevant variables were used to calculate APACHE-IV. Length of stay and death or discharge, Glasgow coma score, and acute physiology score were also evaluated. Moreover, the accuracy of APACHE-IV for mortality was assessed using area under the Receiver Operator Characteristic (ROC) curve. Results: Of the studied patients, 157 died and 682 were discharged (non-survivors and survivors, respectively). The length of stay in the ICU was 10.98±14.60, 10.22 ± 14.21 and 14.30±15.80 days for all patients, survivors, and non-survivors, respectively. The results showed that APACHE-IV model underestimated length of stay in our emergency ICU (p<0.001). In addition, the overall observed mortality was 17.8%, while the predicted mortality by APACHE-IV model was 21%. Therefore, there was an overestimation of predicted mortality by APACHE-IV model, with an absolute difference of 3.2% (p=0.036). Conclusion: The findings showed that APACHE-IV was a poor predictor of length of stay and mortality rate in emergency ICU. Therefore, specific models based on big sample sizes of Iranian patients are required to improve accuracy of predictions.", "keywords": [ "APACHE-IV", "Mortality", "Length of stay", "Intensive care unit", "emergency" ], "content": "Introduction\n\nClinical assessment of disease severity is an important part of medical practice to predict mortality and morbidity in Intensive Care Unit (ICU)1. An acceptable goal in ICU is saving the lives of critically ill patients, since not all patients admitted to an ICU have a normal life after leaving and some will not survive because of disease severity2.\n\nSpecialties of ICUs should predict patient outcomes to focus more on efficient use of ICU beds for critically ill patients2. Disease severity scoring systems can be used as a guidance for clinicians in the objective assessment of disease outcomes and estimation of the chance of recovery2. Acute Physiology And Chronic Health Evaluation (APACHE), introduced in 1981, considers various parameters, including vital signs, physiological variables, neurological score, urine output, age, and comorbid conditions3. The latest version of APACHE-IV is calculated based on 129 variables derived within the first 24 h of ICU admission1, which were assessed from over 110,588 patients admitted to more than 104 ICUs across the USA4,5. Some studies have suggested the superior advantage of APACHE-IV compared to other risk scoring systems6,7.\n\nEvaluation of clinical outcomes and effectiveness of care in ICU patients is influenced by predictive scoring models that compute measures of disease severity and the associated probability of death. APACHE is a logistic regression model involving both physiological and laboratory parameters. It is a commonly used ICU stratification instrument, which is known as an accurate predictor of mortality. Yet, model accuracy decreases over time and requires updating occasionally. A study conducted in 2012 indicated that APACHE-III performance was inadequate even with a predicted mortality of only 2% higher than the observed mortality rate (16% vs. 14%)8. A similar study conducted on APACHE-IV showed that the ICU’s outcome prediction by the model is different to observed values in clinical setting between the predicted and the observed mortality rate9.\n\nTo our knowledge, no study has been conducted to evaluate the accuracy of APACHE-IV for predicting mortality and length of stay in emergency ICUs in Iran. This study aimed to evaluate the hypothesis that the mortality and length of stay in emergency ICUs predicted by APACHE-IV is different than that observed in reality.\n\n\nMethods\n\nThis was a retrospective cohort study conducted on the medical records of 839 consecutive patients admitted in the emergency ICUs in Nemazi Hospital, Shiraz, Iran, between July 2012 and July 2015. The patients of this study were selected from all patients referred to the ICUs of the Center during the study period using convenient sampling method. The total number of patients admitted during this period was 839. The inclusion criterion was minimum 24 hour admission in the ICU and there was no exclusion criterion for this study. The Namazi Hospital is a tertiary referral hospital affiliated to Shiraz University of Medical Sciences, Shiraz, Iran. All the experimental procedures and study protocol of the study were approved by the local ethics committee of Shiraz University of Medical Sciences (protocol no. 94-7636), which were in complete accordance with the ethical standards and regulations of human studies of the Declaration of Helsinki (2014).\n\nThe medical records of 839 consecutive patients admitted to the emergency ICUs of Nemazi Hospital were analyzed. The variables used to calculate APACHE-IV score included age, sex, dates of admission, discharge or death, systolic and diastolic blood pressure, body temperature, heart rate, respiratory rate, glucose, blood urea nitrogen, serum sodium, creatinine, blood hematocrit, white blood cells, serum albumin and bilirubin, urine output during the first 24 h of ICU admission, pH, fraction of inspired oxygen, partial pressure of carbon dioxide, partial pressure of oxygen, and bicarbonate5.\n\nDeath or discharge and length of stay in ICU were followed up by referring to patients’ medical records. Additionally, APACHE-IV score, Glasgow coma score (GCS), and acute physiology score (APS) were calculated according to www.cerner.com (the authors registered as a user in order to calculate all the parameters).\n\nQualitative variables were expressed as number and percentage, and quantitative variables as mean ± standard deviation. Student’s t-test, Mann–Whitney U, Wilcoxon rank test, and Chi-square tests were used where appropriate to compare survivors and non-survivors regarding demographic and clinical variables. In addition, Spearman’s correlation coefficient was used to examine the relationship between APACHE-IV score and length of stay in ICU. Finally, accuracy of APACHE-IV for mortality was assessed using area under the Receiver Operator Characteristic (ROC) curve with an attribution of ‘good’ > 0.80. The data are expressed as mean ± SD for all variables. All statistical analyses were carried out using Stata (version 13, Windows). As the distribution of the quantitative variables was not normal, Mann Whitney U test was used for comparisons of the difference between the survivor and non-survivor groups. For the sex variable the Chi-square test was used. p ≤ 0.05 was considered to be statistically significant.\n\n\nResults\n\nThis study was conducted on 839 patients among whom, 157 died and 682 were discharged (non-survivors and survivors, respectively). The length of stay in ICU was 10.98±14.60, 10.22±14.21, and 14.30±15.80 days in all patients, survivors, and non-survivors, respectively. Demographic information and the clinical features of the patients are summarized in Table 1.\n\nFor the comparisons of quantitative variables Mann Whitney U test was used and for the sex variable the Chi square test was used. P < 0.05 represents significant difference between the survivor and non-survivor groups.\n\nThe results showed no significant difference between the two groups regarding sex (p=0.243). However, the two groups were significantly different with respect to the means of age (p≤0.001), ICU length of stay (p≤0.001), GCS (p≤0.001), APACHE-IV score (p≤0.001), and APS (p≤0.001) (Table 1).\n\nOutcome variables have been summarized in Table 2. Accordingly, mean ± SD of observed length of stay in ICU was 10.98±14.60 days. However, predicted ICU length of stay by the APACHE-IV model was 5.43±2.50 days (p<0.001). This indicated that APACHE-IV underestimated ICU length of stay in our emergency ICU. Additionally, the overall observed mortality was 17.8%, while the predicted mortality by APACHE-IV was 21%. Thus, mortality was overestimated by APACHE-IV model with an absolute difference of 3.2% (p=0.036).\n\nFor the comparisons of stay length Mann Whitney U test was used and for the mortality rate the Chi square test was used. P < 0.05 represents significant difference between the survivor and non-survivor groups.\n\nROC curve for APACHE-IV score and observed mortality has been depicted in Figure 1. Accordingly, area under the curve of the APACHE-IV score was 0.81, 95% CI (0.77, 0.84). These values were statistically significant and could be an appropriate predictor for observed mortality. Nevertheless, there was a significant weak correlation between APACHE-IV score and observed ICU length of stay (r=0.175, p<0.0001).\n\n\nDiscussion\n\nA retrospective cohort study was conducted among 839 patients referred to ICU at Namazi Hospital in Shiraz, Iran. The study results showed that APACHE-IV underestimated the length of stay in our emergency ICU. In addition, the overall observed mortality was 17.8%, while the predicted mortality by APACHE-IV was 21%. Thus, there was an overestimation of predicted mortality by APACHE-IV, with an absolute difference of 3.2% (p=0.036).\n\nSeveral factors may contribute to poor performance of APACHE-IV in emergency ICU. APACHE-IV is a good benchmark to determine disease severity; however, the present study results indicated that it did not function well to predict the risk of mortality and length of stay in emergency ICU. Other studies also reported this score not to be predictive of mortality10,11. The poor estimate may be attributed to various reasons. Firstly, the estimations were achieved based on American rather than our own patients’ data. Generally, predictive scoring systems function appropriately in populations where scores are derived from the same population data. Therefore, many experts recommend external validation at national, regional, or institutional levels. For example, APS3 has several customized versions for seven geographic regions12,13.\n\nSecondly, in America, where APACHE was calibrated, patients go from ICU to ‘step down’, a halfway ward, before moving to general wards. In Iran, patients directly go to general wards, and consequently, they have to stay in ICUs for a longer time period than American patients.\n\nThirdly, even if scores are achieved by patients’ data, they must be calibrated over time. This is because case-mix varies, quality of care improves, and types of disease changes over time. In general, accurate calibration is a key characteristic that should be ensured for all risk scoring systems. Calibration may weaken over time, especially due to the effects of altered patient interventions and case-mix. This often results in overestimation of death or mortality14.\n\nThe findings of the present study revealed that APACHE-IV score based on our data would be an appropriate predictor for the observed mortality, while this relationship was not confirmed by the APACHE-IV score according to the American database9. Moreover, our findings showed a similar relationship between APACHE-IV score and ICU length of stay with the study conducted on the United States database9. Overall, a large patient’s database should exist in order for APACHE-IV to correctly predict outcomes (i.e. mortality and ICU length of stay).\n\nThe strength of this study should be noted. This study is the first study in Iran that demonstrated that predictions of mortality and ICU length of stay should be based on data obtained from Iranian and not American patients. However, this study had some limitations, the first of which being the intrinsic shortcomings of its retrospective design (inability to confirm causation, and dependence on medical records). Another study limitation was its small sample size; however, to date, our cohort of 839 patients is the largest reported study of patients admitted to emergency ICUs in Iran.\n\nIn conclusion, the findings of this study suggested that the American based APACHE-IV score is a poor predictor of length of stay and mortality in emergency ICU in Iran. Therefore, specific models based on big sample sizes of our patients from Iran are required to improve the accuracy of predictions of mortality and ICU length of stay for our country.\n\n\nData availability\n\nDataset 1: Data for the study on efficacy of APACHE-IV for predicting mortality and length of stay in an intensive care unit in Iran. doi, 10.5256/f1000research.12290.d17798715", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis study was financially supported by Shiraz University of Medical Sciences (grant number 94-7636).\n\n\nAcknowledgements\n\nThe present article was extracted from the thesis written by Mohammad Ghorbani. The authors would like to thank Ms. A. Keivanshekouh at the Research Improvement Center of Shiraz University of Medical Sciences for improving the use of English in the manuscript.\n\n\nReferences\n\nSaleh A, Ahmed M, Sultan I, et al.: Comparison of the mortality prediction of different ICU scoring systems (APACHE II and III, SAPS II, and SOFA) in a single-center ICU subpopulation with acute respiratory distress syndrome. Egyptian Journal of Chest Diseases and Tuberculosis. 2015; 64(4): 843–8. Publisher Full Text\n\nRahimzadeh P, Taghipur Anvari Z, Hassani V: Estimation of mortality rate of patients in surgical intensive care unit of Hazrat-Rasul hospital of Tehran using the APACHE II standard disease severity scoring system. Hakim Res J. 2008; 11(1): 22–8. Reference Source\n\nKnaus WA, Zimmerman JE, Wagner DP, et al.: APACHE-acute physiology and chronic health evaluation: a physiologically based classification system. Crit Care Med. 1981; 9(8): 591–7. PubMed Abstract \| Publisher Full Text\n\nZimmerman JE, Kramer AA, McNair DS, et al.: Intensive care unit length of stay: Benchmarking based on Acute Physiology and Chronic Health Evaluation (APACHE) IV. Crit Care Med. 2006; 34(10): 2517–29. PubMed Abstract \| Publisher Full Text\n\nZimmerman JE, Kramer AA, McNair DS, et al.: Acute Physiology and Chronic Health Evaluation (APACHE) IV: hospital mortality assessment for today's critically ill patients. Crit Care Med. 2006; 34(5): 1297–310. PubMed Abstract \| Publisher Full Text\n\nKuzniewicz MW, Vasilevskis EE, Lane R, et al.: Variation in ICU risk-adjusted mortality: impact of methods of assessment and potential confounders. Chest. 2008; 133(6): 1319–27. PubMed Abstract \| Publisher Full Text\n\nBrinkman S, Bakhshi-Raiez F, Abu-Hanna A, et al.: External validation of Acute Physiology and Chronic Health Evaluation IV in Dutch intensive care units and comparison with Acute Physiology and Chronic Health Evaluation II and Simplified Acute Physiology Score II. J Crit Care. 2011; 26(1): 105.e11–8. PubMed Abstract \| Publisher Full Text\n\nPaul E, Bailey M, Van Lint A, et al.: Performance of APACHE III over time in Australia and New Zealand: a retrospective cohort study. Anaesth Intensive Care. 2012; 40(6): 980–94. PubMed Abstract\n\nZimmerman JE, Kramer AA: Outcome prediction in critical care: the Acute Physiology and Chronic Health Evaluation models. Curr Opin Crit Care. 2008; 14(5): 491–7. PubMed Abstract \| Publisher Full Text\n\nHanisch E, Brause R, Paetz J, et al.: Review of a large clinical series: Predicting death for patients with abdominal septic shock. J Intensive Care Med. 2011; 26(1): 27–33. PubMed Abstract \| Publisher Full Text\n\nChan T, Bleszynski MS, Buczkowski AK: Evaluation of APACHE-IV Predictive Scoring in Surgical Abdominal Sepsis: A Retrospective Cohort Study. J Clin Diagn Res. 2016; 10(3): Pc16–8. PubMed Abstract \| Free Full Text\n\nMetnitz PG, Moreno RP, Almeida E, et al.: SAPS 3--From evaluation of the patient to evaluation of the intensive care unit. Part 1: Objectives, methods and cohort description. Intensive Care Med. 2005; 31(10): 1336–44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoreno RP, Metnitz PG, Almeida E, et al.: SAPS 3--From evaluation of the patient to evaluation of the intensive care unit. Part 2: Development of a prognostic model for hospital mortality at ICU admission. Intensive Care Med. 2005; 31(10): 1345–55. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKelley MA, Manaker S, Finlay G: Predictive scoring systems in the intensive care unit. UpToDate. 2012. Reference Source\n\nGhorbani M, Ghaem H, Rezaianzadeh A, et al.: Dataset 1 in: A study on the efficacy of APACHE-IV for predicting mortality and length of stay in an intensive care unit in Iran. F1000Research. 2017. Data Source" }	[ { "id": "28177", "date": "22 Nov 2017", "name": "Nelson Elias", "expertise": [ "Reviewer Expertise Orthopedics callus formation", "bone growth and biodegradable implants" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nClassification: Research - retrospective cohort study with a critical review of the corresponding literature in Apache IV scoring systems\n\nOriginality and Relevance: a)\n\nPertinent for the journal - F1000Research b)\n\nOriginal c)\n\nContribution towards the development of specific models based in the characteristics of Iranian population\n\nContent a\n\nCoherence between the paper’s title and its content b\n\nCoherence between the paper’s summary and its content c\n\nThe article fulfils its stated objectives d\n\nThe conclusion is adequate e\n\nRelevant and current bibliographic references (I didn’t find in the text the reference 15)\n\nThe discussion of the topic is ordered, didactic, clear and concise\n\nThe article should be published in the journal with slight modification (include reference 15 in the text)\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "28373", "date": "27 Nov 2017", "name": "Ali Yadollahpour", "expertise": [ "Reviewer Expertise Experimental neurology", "medical physics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe manuscript entitled \"A study on the efficacy of APACHE-IV for predicting mortality and length of stay in an intensive care unit in Iran\" was reviewed. This is a well conducted study with appropriate methodology. The data provided are of good volume and with enough details. The sample size is of particularly important resulting in the more robust evidence. The authors could appropriately discussed the results and compared their findings with the similar conducted studies. Conducting further studies in the country with big sample size in the ICU patients can shed further lights to adopt appropriate policies for improving the healthcare conditions and reducing the mortality rate of ICU patients.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2032
https://f1000research.com/articles/6-997/v1	26 Jun 17	{ "type": "Software Tool Article", "title": "RNAtor: an Android-based application for biologists to plan RNA sequencing experiments", "authors": [ "Shruti Kane", "Himanshu Garg", "Neeraja M. Krishnan", "Aditya Singh", "Binay Panda", "Shruti Kane", "Himanshu Garg", "Neeraja M. Krishnan", "Aditya Singh" ], "abstract": "RNA sequencing (RNA-seq) is a powerful technology that identifies novel transcripts (coding, non-coding and splice variants), understands transcript structures, and estimates gene/allele expression. Biologists face specific challenges while designing RNA-seq experiments. The nature of these challenges lies in determining the total number of sequenced reads and replicates required for detecting marginally differentially expressed transcripts, and in determining the adequate number of lanes to use in a sequencing flow cell. Despite previous attempts to address these challenges, easily accessible and biologist-friendly mobile applications do not exist. Thus, we developed RNAtor, a mobile application for Android platforms, to aid biologists in correctly designing their RNA-seq experiments. The recommendations from RNAtor are based on simulations and real data.", "keywords": [ "RNA-seq", "Android-based", "simulations", "mobile application", "recommendations", "experimental design" ], "content": "Introduction\n\nRNA-seq offers several advantages over low-throughput technologies such as quantitative PCR and annotation-dependent methods such as microarrays. Designing RNA-seq experiments accurately, however, poses challenge to biologists. This is particularly true when prior knowledge on genome or transcriptome of the organism of choice is not available. It is important to determine the number of replicates and the number of sequencing reads, and choose the right analytical tool, to estimate subtle differences between expression levels of transcripts. In the current manuscript, we describe RNAtor, an Android app with a user-friendly graphical user interface (GUI) that helps biologists design RNA-seq experiments. RNAtor can be linked to any existing differential expression analysis tool, and can help design experiments to estimate expression differences of as low as 0.8–1.2X fold. RNAtor’s recommendations are based on an exhaustive combination of discovery with simulated reads for transcriptomes of varying sizes (3 to 100 MB), followed by validation with real datasets on wild-type and mutant conditions of Saccharomyces cerevisiae.\n\n\nMethods\n\nWe simulated varying numbers of Illumina-like reads with replicates, with fold changes ranging from 1.2–5X between the control and treatment samples on a 3 MB human chr14 (hg19) transcriptome, using Polyester (Frazee et al., 2015). We detected differentially expressed genes (DEGs) on all the simulations using Tophat v2.1.1-Cufflinks v2.2.1 (Trapnell et al., 2012) based genome-guided workflow followed by differential expression analyses using five tools: Deseq v1.28.0 (Anders & Huber, 2010); Deseq2 v1.16.1 (Love et al., 2014); EdgeR v3.18.1 (Robinson et al., 2010); Cuffdiff-Cufflinks v2.2.1 (Trapnell et al., 2012); and Kallisto v0.43.1 (Bray et al., 2016) and a de novo assembly-based tool, Trinity v2.3.2 (Grabherr et al., 2011) followed by differential expression analyses using Kallisto v0.43.1 (Bray et al., 2016). We studied results from these simulations on the number of DEGs detected reliably and the extent of recovery of those DEGs. Based on these simulations, we arrived at recommendations on the number of reads, number of replicates, and the tool(s) needed to identify DEGs reliably. We validated these recommendations using simulated reads from larger transcriptomes (10MB, 30MB and 100MB), created by combining transcriptomes from more than one hg19 chromosome, and using a real Sacharomyces cerevisiae dataset (ENA accession: ERP004763) comprising of 48 biological replicates, for two conditions; wild-type (WT) and a snf2 knock-out (KO) mutant.\n\nThe size of the transcriptome (or genome if the transcriptome size is not known), taken from a user-defined or from a backend database, the number of replicates to use and the fold change of DEGs are user-defined parameters in RNAtor (Figure 1). An RNAtor flowchart highlighting simulation conditions and analytical tools used is provided in Supplementary Figure S1.\n\n\nResults\n\nRNAtor was evaluated using questions that a biologist would typically ask before starting an experiment, followed by the recommendations provided by RNAtor.\n\n1, 1.5, 6, 10, 14 and 20 million reads are needed for detection of differential expression of DEGs at 5-fold, 4-fold, 3-fold, 2-fold, 1.5-fold and 1.2-fold change, respectively, for a 3Mb transcriptome with 3 replicate samples.\n\nWe simulated 0.2–20 million reads for human chromosome 14 (~3Mb) and observed that the numbers of detected DEGs simulated at a given fold change peaked for a certain coverage before plateauing (Figure 2). This observation remained valid for the real data (Figure 3) and the large simulated transcriptomes (10Mb, 30Mb and 100Mb) (Supplementary Figure S2). Increasing the number of sequencing reads increased the sensitivity of detection. The final recommendations from RNAtor correspond to the number of DEGs at its peak, and are therefore a good compromise between sensitivity and keeping the cost of sequencing low. Changing the number of replicates does change the recommendation. For example, with more than three replicates, RNAtor suggests producing fewer reads to obtain the same information (Table 1).\n\nKallisto detected optimal number of DEGs with the highest sensitivity. Focusing purely on the number of DEGs detected between WT and KO, Kallisto performed best over the other tools tested (Figure 2 and Supplementary Figure 3).\n\nCuffdiff can be used for high specificity and DeSeq2 and EdgeR, for high transcript recovery. Although Kallisto-Sleuth was fast and produced results with high sensitivity; we observed that this was at the expense of specificity of detection (Supplementary Figure S3). Cuffdiff produced results with high specificity albeit with a loss of sensitivity (Supplementary Figure S3). The transcript recovery was best for EdgeR among the 3 tools tested (CuffDiff, DeSeq and EdgeR, Supplementary Figure S4).\n\nThe assembly-based pipeline yields more DEGs with higher specificity. Using Trinity (Grabherr et al., 2011) as an assembly pipeline along with Kallisto enhanced the number of DEGs detected when compared with the genome-guided Kallisto-Sleuth pipeline (Figure 2). The specificity of Trinity-Kallisto was also better when compared to the Kallisto-Sleuth pipeline (Supplementary Figure S3).\n\n\nDiscussion\n\nAlthough some of the challenges with RNA-seq experiments have been addressed previously (Busby et al., 2013; Luo et al., 2014), currently there is no easy-to-use, biologist-friendly mobile phone-based app. Scotty, a previously reported, useful, interactive web-based tool aids RNA-seq experimental design. However, it has a dependence on pilot or prototype data, closely matching the actual experimental conditions (Busby et al., 2013). Additionally, it can detect genes or transcripts of only up to 2X fold change in the test condition relative to the control. RNAtor addresses some of these gaps as a user-friendly mobile app, however it has certain limitations. For example, it does not take into account the dynamic nature of any transcriptome (where the exact size of transcriptome is not known and cannot simply be derived from the genome size), the throughput of different sequencing instruments and the presence of spliced variants. These limitations will be addressed in future releases.\n\n\nData availability\n\nThe Android version of RNAtor is available on Google Play Store.\n\nLatest source code: https://github.com/binaypanda/RNAtor.\n\nArchived source code as at the time of publication: https://doi.org/10.5281/zenodo.814905 (Panda, 2017).\n\nLicense: RNAtor v1.0 is distributed under GNU GPLv3 licence.", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nResearch presented in this article is funded by the Department of Electronics and Information Technology, Government of India (Ref No: 18(4)/2010-E-Infra., 31-03-2010) and Department of IT, BT and ST, Government of Karnataka, India (Ref No: 3451-00-090-2-22).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary Figure S1: RNAtor flowchart highlighting simulation conditions (reads, replicates, and fold change of differential expression) and analytical tools used.\n\nClick here to access the data.\n\nSupplementary Figure S2: Number of differentially expressed genes (DEGs) detected for various simulated dataset on 10Mb, 30Mb and 100Mb transcriptomes using the Kallisto-Sleuth pipeline.\n\nClick here to access the data.\n\nSupplementary Figure S3: True/false positive curves for differentially expressed genes (DEGs) recovered under various simulation conditions, created by combining reads (0.1M–20M), replicates (2–5) and fold change of differential expression (1.2–5X) by Cuffdiff, Deseq2, EdgeR, Kallisto and Trinity-Kallisto tools.\n\nClick here to access the data.\n\nSupplementary Figure S4: Percentage recovery of transcripts under various simulation conditions, created by combining reads (0.1M–20M), replicates (0–5) and fold change of differential expression (1.2–5X) with CuffDiff, DeSeq and EdgeR. The size of the bubble represents the extent of transcript recovery.\n\nClick here to access the data.\n\n\nReferences\n\nAnders S, Huber W: Differential expression analysis for sequence count data. Genome Biol. 2010; 11(10): R106. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBray NL, Pimentel H, Melsted P, et al.: Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol. 2016; 34(5): 525–527. PubMed Abstract \| Publisher Full Text\n\nBusby MA, Stewart C, Miller CA, et al.: Scotty: a web tool for designing RNA-Seq experiments to measure differential gene expression. Bioinformatics. 2013; 29(5): 656–657. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFrazee AC, Jaffe AE, Langmead B, et al.: Polyester: simulating RNA-seq datasets with differential transcript expression. Bioinformatics. 2015; 31(17): 2778–2784. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGrabherr MG, Haas BJ, Yassour M, et al.: Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29(7): 644–652. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove MI, Huber W, Anders S: Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15(12): 550. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLuo H, Li J, Chia BK, et al.: The importance of study design for detecting differentially abundant features in high-throughput experiments. Genome Biol. 2014; 15(12): 527. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nPanda B: binaypanda/RNAtor: RNAtor. 2017. Data Source\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–140. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTrapnell C, Roberts A, Goff L, et al.: Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012; 7(3): 562–578. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "24054", "date": "06 Jul 2017", "name": "Daisuke Komura", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors developed a new mobile application named RNAtor to assist in the designing of RNA-seq experiments. It provides users with the number of reads that is required for optimal detection of differentially expressed genes at a given fold-change threshold based on simulations and real data.\nThis is a useful tool for NGS users. However there are some errors and suggestions that need to be fixed.\n\nMajor:\nIn general, some reads in RNA-seq analysis are removed due to their low sequencing quality or cannot be mapped probably due to library contamination of rRNA or other species’ RNA. Does this software consider the effect? If not, it could underestimate the number of required reads.\n\nHow does RNAtor calculate the number of reads required for DEG detection? The authors claim that it is calculated based on the number of DEGs at its peak in both real and simulated data but the exact algorithm is not shown. Specifically, how were the peak values calculated and how were the peak values of real and simulated data merged?\n\nMinor:\n\nWhich does the term ‘replicates’ mean in this manuscript, technical replicate or biological replicate?\n\nImplementation: “… workflow followed by differential expression analysis using five tools:…” I think four instead of five is correct because Kallisto does not use outputs of Tophat-Cufflinks pipeline (Supplementary Figure 1).\n\nFigure 2: How many DEGs were included in total in the simulated datasets? Sensitivity (%) is preferable to the number of DEGs in this case. The number of replicate is a discrete value but the slope in this figure is smooth. What do the numbers in fold change (1.2 0.83-5, 0.2) mean?\n\nFigure 3: Why were the result of 5x and 4x merged? What does each line indicate? # reads = 0 means nothing thus should be removed.\n\nIs the rationale for developing the new software tool clearly explained? Partly\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3175", "date": "16 Nov 2017", "name": "Binay Panda", "role": "Author Response", "response": "We thank Dr. Daisuke Komura for his time and timely review. We have addressed all his queries below and have incorporated his suggestions in the revised version of the manuscript, which we believe is much improved now. Response to Reviewers’ comments Major: Reviewer’s comments: 1) In general, some reads in RNA-seq analysis are removed due to their low sequencing quality or cannot be mapped probably due to library contamination of rRNA or other species’ RNA. Does this software consider the effect? If not, it could underestimate the number of required reads. Authors’ response: No, the software does not consider this effect. The simulated reads correspond to error-free/-corrected reads. The add_error and add_platform_error features are added in a recent release of Polyester, not available when we used Polyester. Reviewer’s comments: 2) How does RNAtor calculate the number of reads required for DEG detection? The authors claim that it is calculated based on the number of DEGs at its peak in both real and simulated data but the exact algorithm is not shown. Specifically, how were the peak values calculated and how were the peak values of real and simulated data merged? Authors’ response: The detected DEGs by various tools were plotted as a function of simulated reads and replicates (Figure 2). The saturation point of the DEG curves, i.e. the peak was used to recommend a certain no of reads given the no of replicates, to detect optimal DEGs. Validation for this recommendation was obtained from extrapolation of the read numbers according to the Saccharomyces transcriptome size (Figure 3). Minor: Reviewer’s comments: 3) Which does the term ‘replicates’ mean in this manuscript, technical replicate or biological replicate? Authors’ response: We thank the reviewer to point this out. The replicates used were technical. We have made this change in the revised manuscript. Reviewer’s comments: 4) Implementation: “… workflow followed by differential expression analysis using five tools:…” I think four instead of five is correct because Kallisto does not use outputs of Tophat-Cufflinks pipeline (Supplementary Figure 1). Authors’ response: Kallisto is used twice; first, with the genome-guided paradigm and second, with de novo assembly using Trinity. In the first scenario, the Tophat-Cufflinks alignments (.bam) were converted to reads (.fastq) to be used with Kallisto along with the 3MB transcriptome as the reference. In the second scenario, the de novo assembled transcriptome was used as the reference along with the simulated reads with Kallisto. This has been clarified in the revised manuscript. Reviewer’s comments: 5) Figure 2: How many DEGs were included in total in the simulated datasets? Sensitivity (%) is preferable to the number of DEGs in this case. The number of replicate is a discrete value but the slope in this figure is smooth. What do the numbers in fold change (1.2 0.83-5, 0.2) mean? Figure 2: I think \"fold change (1.2,0.83 - 5,0.2)\" is confusing so it might be better to change it to another one such as ((>1.2 or <1/1.2) to (>5 or <1/5)). Authors’ response: The total no of simulated DEGs are 363. TP and FP curves are represented in Supplementary Figure S3. We have changed the representation for replicate numbers. The simulated fold changes were 1.2X to 5X, in both directions, i.e. 1.2,1/1.2=0.83 and 5,1/5=0.2. Fold change (1.2,0.83 - 5,0.2)\" is changed to ((>1.2 or <1/1.2) to (>5 or <1/5)). Reviewer’s comments: 6) Figure 3: Why were the result of 5x and 4x merged? What does each line indicate? # reads = 0 means nothing thus should be removed. Figure 3: What do the lines indicate in each subplot? Authors’ response: The number of DEGs obtained separately at 4X and 5X were not sufficient to draw any conclusions, Hence they were merged as 4X+5X or >4X. We have removed the #reads=0 data point. We have updated the Figure 3 legend." } ] }, { "id": "23782", "date": "30 Aug 2017", "name": "Niranjan Nagarajan", "expertise": [ "Reviewer Expertise Genomics", "Computational Biology" ], "suggestion": "Not Approved", "report": "Not Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nRNAtor looks like a simple and easy to use application. However this could also be its drawback in that it may be too simplistic. The authors should show some evidence that the parts they omit do not significantly impact RNAtor's guidance to users.\nAbstract:\n1) Perhaps the first sentence of the abstract should be reworded as it is a bit odd to say that \"RNA Sequencing ... understands transcript structures\" etc.\n2) Its unnecessary to mention the \"number of lanes\" when the \"number of reads\" has been highlighted.\n3) It is s not clear why a mobile application is necessary. Is a web-based tool like EDDA not sufficient?\nIntroduction:\n4) The last sentence is not very clear in terms of what is being done here.\n5) As it is typical to present and discuss prior work in the introduction, I believe it is important to add this component. Some of this could perhaps come from the current discussion section.\nImplementation:\n6) What were the parameters used for running Polyester? In particular, different experimental conditions will likely have different expression profiles, biological variability dictating the dispersion parameters and perhaps other factors like splicing complexity, gene families etc. that all affect RNA-seq analysis. How does RNAtor account for these? How were the various differential analysis softwares run? They usually have cutoffs that can be chosen to make their results more or less stringent.\nResults:\n7) The detection of differentially abundant RNAs using RNA-seq is impacted by the relative abundance of the transcript. How is this taken into account in table 1?\n8) In figure 2, are these all true positives? What about false positives? Usually there is a tradeoff and a user needs to be aware of this.\n9) Figure 3 does not have a legend.\n10) It is not clear to me what transcript recovery is referring to and why that is relevant here.\n11) Supplementary figure 4 needs better resolution and font sizes.\n12) The claims made in the section \"Detection specificity and transcript recovery by DE tools\" are not obvious from the figures shown.\nDiscussion:\n13) It seems to me that there are many more limitations in RNAtor than are discussed here. Also, it is appropriate to discuss the strengths and weaknesses of EDDA as well and perhaps some of this should be in the introduction, as noted earlier.\n\nIs the rationale for developing the new software tool clearly explained? No\n\nIs the description of the software tool technically sound? No\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? No", "responses": [ { "c_id": "3174", "date": "16 Nov 2017", "name": "Binay Panda", "role": "Author Response", "response": "We thank Dr. Niranjan Nagarajan for reviewing the manuscript and providing his comments. Following his suggestions, we have revised the manuscript and have responded to all his queries below. We believe this has improved the manuscript. Reviewer’s comments: RNAtor looks like a simple and easy to use application. However this could also be its drawback in that it may be too simplistic. The authors should show some evidence that the parts they omit do not significantly impact RNAtor's guidance to users. Authors’ response: We agree with the reviewer that the RNAtor v1.0 is simplistic and some of the assumptions can affect the recommendations. However, the tool is intended to be used by biologists who use RNA-seq as a replacement to existing technology like microarray and qPCR, to ask simpler biological questions. Many small biology labs, we believe, are still in this group. The tool is not intended to serve advanced users. We are well aware of its limitations and intend to expand the scope of the tool in its future versions, by introducing biases that mimick various experimental conditions into the simulation phase. Nevertheless, we believe that the validation of the recommendations resulting from training on simulated RNA-seq data that has not yet incorporated various biological biases, with real data from Saccharomyces cerevisiae provides strong evidence that our assumptions do not significantly impact RNAtor's guidance to users. Abstract: Reviewer’s comments: 1) Perhaps the first sentence of the abstract should be reworded as it is a bit odd to say that \"RNA Sequencing ... understands transcript structures\" etc. Authors’ response: We thank the reviewer to point this. We have re-worded this sentence in the revised manuscript. Reviewer’s comments: 2) Its unnecessary to mention the \"number of lanes\" when the \"number of reads\" has been highlighted. Authors’ response: Following the reviewer’s suggestion, we have removed the use of the term in the revised manuscript. Reviewer’s comments: 3) It is s not clear why a mobile application is necessary. Is a web-based tool like EDDA not sufficient? Authors’ response: We agree with the reviewer that there are web-based tools, like EDDA and Scotty, which we have now elaborated in the revised manuscript, that can do similar job. However, we strongly believe that a mobile application offers a lot more flexibility, ease of navigation, and user-friendliness, compared to a web-based tool. Besides, many features of the App are available for offline use and the user has the flexibility to use it on the work bench or anywhere convenient. Additionally, Apps use a different framework to run code and hence, run faster than mobile websites that typically use javascript to run their code, and store the data locally on the mobile device, unlike websites that store data on web servers. Introduction: Reviewer’s comments: 4) The last sentence is not very clear in terms of what is being done here. Authors’ response: Following the reviewer’s suggestion, we have modified the sentence in the revised manuscript. Reviewer’s comments: 5) As it is typical to present and discuss prior work in the introduction, I believe it is important to add this component. Some of this could perhaps come from the current discussion section. Authors’ response: We have now modified the introduction to include references on tools aiding RNA-seq experimental design. Implementation: Reviewer’s comments: 6) What were the parameters used for running Polyester? In particular, different experimental conditions will likely have different expression profiles, biological variability dictating the dispersion parameters and perhaps other factors like splicing complexity, gene families etc. that all affect RNA-seq analysis. How does RNAtor account for these? How were the various differential analysis softwares run? They usually have cutoffs that can be chosen to make their results more or less stringent. Authors’ response: The per_reads_transcript, num_reps, fold_changes, in addition to the input ‘fasta’ and output ‘outdir’ parameters were exercised in polyester simulations. All differential expression analysis softwares were run with default cut-offs. We have now mentioned these in the revised manuscript Results: Reviewer’s comments: 7) The detection of differentially abundant RNAs using RNA-seq is impacted by the relative abundance of the transcript. How is this taken into account in table 1? Authors’ response: In the current version of the tool implementation, the simulated data does not take into account the relative abundance of transcript, for e.g. in relation to a control gene or any other gene of interest. Hence, our recommendations (Table 1) are not affected by these. However, differences in relative abundances of true control genes between treatment and control would lead to over- or under-estimation of DEGs, and therefore, the base-line assumption of log2FC=0 should be accordingly adjusted. We have acknowledged this limitation in the revised manuscript. Reviewer’s comments: 8) In figure 2, are these all true positives? What about false positives? Usually there is a tradeoff and a user needs to be aware of this. Authors’ response: Figure 2 are all detected DEGs, i.e., both true and false positives. Supplementary Figure S3 gives the true positive and false positive trends separately. Yes, there is a tradeoff between sensitivity (true postives) and specificity (absence of false positives). Kallisto performs with a high sensitivity while compromising on specificity, while CuffDiff performs with a high specificity while compromising on sensitivity. We have elaborated this in the revised manuscript. Reviewer’s comments: 9) Figure 3 does not have a legend. Authors’ response: We thank the reviewer to point this out and have now added a legend to Figure 3. Reviewer’s comments: 10) It is not clear to me what transcript recovery is referring to and why that is relevant here. Authors’ response: Transcript recovery refers to the length of the transcript as assembled by Tophat, detected as differentially expressed by EdgeR or CuffDiff or DESeq2, in relation to the actual length, as per simulations. It is possible to estimate this parameter only for these three tools, since they offer a handle to the actual transcript Ids. This has been clarified in the revised manuscript. Reviewer’s comments: 11) Supplementary figure 4 needs better resolution and font sizes. Authors’ response: Supplementary Figure 4 with improved resolution has now been added. Reviewer’s comments: 12) The claims made in the section \"Detection specificity and transcript recovery by DE tools\" are not obvious from the figures shown. Authors’ response: The results in this section have been elaborated further with reference to Supplementary Figure S4 in the revised manuscript. With reference to the claim made about detection specificity citing Supplementary Figure S3, we found that CuffDiff performs with high specificity at the loss of sensitivity, with the opposite being true for Kallisto-Sleuth." } ] } ]	1	https://f1000research.com/articles/6-997
https://f1000research.com/articles/6-2015/v1	15 Nov 17	{ "type": "Research Article", "title": "IL-15 enhances cross-reactive antibody recall responses to seasonal H3 influenza viruses in vitro", "authors": [ "Junqiong Huang", "Shannon P. Hilchey", "Jiong Wang", "Jessica Gerigan", "Martin S. Zand", "Junqiong Huang", "Shannon P. Hilchey", "Jiong Wang", "Jessica Gerigan" ], "abstract": "Background: Recently, several human monoclonal antibodies that target conserved epitopes on the stalk region of influenza hemagglutinin (HA) have shown broad reactivity to influenza A subtypes. Also, vaccination with recombinant chimeric HA or stem fragments from H3 influenza viruses induce broad immune protection in mice and humans. However, it is unclear whether stalk-binding antibodies can be induced in human memory B cells by seasonal H3N2 viruses. Methods: In this study, we recruited 13 donors previously exposed to H3 viruses, the majority (12 of 13) of which had been immunized with seasonal influenza vaccines. We evaluated plasma baseline strain-specific and stalk-reactive anti-HA antibodies and B cell recall responses to inactivated H3N2 A/Victoria/361/2011 virus in vitro using a high throughput multiplex (mPlex-Flu) assay. Results: Stalk-reactive IgG was detected in the plasma of 7 of the subjects. Inactivated H3 viral particles rapidly induced clade cross-reactive antibodies in B cell cultures derived from all 13 donors. In addition, H3 stalk-reactive antibodies were detected in culture supernatants from 7 of the 13 donors (53.8%). H3 stalk-reactive antibodies were also induced by H1 and H7 subtypes. Interestingly, broadly cross-reactive antibody recall responses to H3 strains were also enhanced by stimulating B cells in vitro with CpG2006 ODN in the presence of IL-15. H3 stalk-reactive antibodies were detected in CpG2006 ODN + IL-15 stimulated B cell cultures derived from 12 of the 13 donors (92.3%), with high levels detected in cultures from 7 of the 13 donors. Conclusions: Our results demonstrate that stalk-reactive antibody recall responses induced by seasonal H3 viruses and CpG2006 ODN can be enhanced by IL-15.", "keywords": [ "Influenza immunity", "B Cell", "CpG ODN", "IL-15", "hemagglutinin stalk" ], "content": "Introduction\n\nWorldwide, annual influenza epidemics are estimated to result in about 3 to 5 million cases of severe illness, and about 250,000 to 500,000 deaths1,2. Preventive vaccination is the major intervention currently used to prevent influenza infections3,4, and is designed to elicit IgG antibodies directed against the hemagglutinin viral surface protein (HA). Current vaccine formulations elicit a potent immune response against viruses that are closely matched to the vaccine strain, largely through targeting epitopes on the globular head of HAs of influenza A H1N1 and H3N2 subtypes and influenza B strains. However, antigenic drift of influenza virus, which is caused by an accumulation of point mutations within the HA sequences, frequently occurs in influenza A strains and this is particularly true for H3 influenza A strains5–7. Individuals who were infected by or vaccinated against H3 influenza viruses circulating in prior years may thus be susceptible to new viral strains.\n\nSince jumping species to humans in 1968, H3N2 swine flu viruses have been responsible for several seasonal pandemics, resulting in both prolonged duration of the influenza season and greater disease severity8,9. Under the selective pressure of host immunity, H3N2 influenza virus HAs have undergone progressive antigenic drift. This became particularly problematic during the 2014–2015 influenza season, when H3N2 strains became predominant and were antigenically and genetically distinct from the A/Texas/50/2012 (A/Tex12) vaccine strain10,11. The resulting antigenic mismatch between the vaccine strain and circulating H3 viruses, lead to extremely low vaccine effectiveness in the northern hemisphere12.\n\nIn contrast to the variable HA head domain, epitopes within the HA stalk domain are highly conserved and have become a main target for development of novel treatments using either antibody-based vaccine design or passive immunotherapy. Several human monoclonal antibodies that target highly conserved epitopes on the stalk region of influenza HA display broad reactivity with group 1 and/or group 2 viruses and protect against lethal challenge with influenza viruses in vivo13–16. Animals immunized against H3 stalk elicited broadly cross-reactive antibodies, resulting in protection from challenges with viruses that are of the same HA subtype and/or group17–19. Others have found that vaccination with a divergent hemagglutinin can increase the frequency of B cells encoding broad influenza A-neutralizing antibodies20. However, stalk-reactive antibodies are rarely found in individuals vaccinated with traditional in-activated influenza virus seasonal vaccines21–23. Recent studies have detected broadly cross-reactive, anti-stalk IgG antibodies in people vaccinated with the pandemic H1N1 A/California/07/09 (pdm A/Cali09) strain24,25. Recombinant chimeric HA from H3 viruses have also been shown to elicit broad immune protection in mice and humans26,27. However, it is unclear whether stalk-binding antibodies can be induced in human memory B cells using seasonal H3N2 viruses.\n\nSeveral strategies have been employed in an attempt to improve broadly cross-reactive IgG production by application of a non-specific stimulus, the most common of which is the addition of various adjuvants to promote increased antibody secretion. In addition, the application of various cytokines has also been studied to increase antibody production, including IL-15. IL-15, a member of the 4-α-helix bundle family of cytokines, signals via hetero-trimeric receptors involving the IL-2 receptor β chain (IL2Rβ ), a common γ chain (IL2Rγc), which is also required for signaling by IL-2, IL-4, IL-7, IL-9 and IL-21, and a unique α subunit (IL-15Rα) that confers receptor specificity to IL-1528. Some cytokines that signal through the common IL-2Rγ chain have been shown to increase activated naive and memory B cell IgG secretion rates29. IL-15 signals through the activation of JAK2, p38 and ERK1/2 MAPK, SYK kinase and the NF-kB transcriptional factor30. Due to the common γc and β chain, IL-15 shares certain functions with IL-2, including T cell proliferation, the generation of cytotoxic T cells, immunoglobulin synthesis by B cells and the generation and persistence of NK cells31,32. IL-15 has been shown to play an essential role in the proliferation of memory B cells and Ig production in vivo33.\n\nIn addition to promoting the proliferation, differentiation, and IgG secretion of germinal center B cells, IL-15 is also involved in the generation and maintenance of long-term serologic memory29,34,35. IL-15 adjuvant has been reported to increase IgG production in animals immunized with influenza vaccines36, and DIII antigens of Japanese encephalitis virus and West Nile virus37. IL-15 adjuvanted immunization with a DNA vaccine comprised of the N1 and NP genes from the H5N1 influenza virus induced early and high antibody response in chickens38. In addition, IL-15 participates in the homing of immature B cells and maintenance of the B cell repertoire39. Finally, IL-15 signaling appears to be essential to CD4 T cell and B cell activation by CpG ODN signaling through TLR940, suggesting further synergy between existing vaccine adjuvants and IL-15.\n\nAs a vaccine adjuvant, IL-15 has been used for the HIV vaccine and cancer trails (www.clinicaltrials.gov: NCT00775424, NCT00115960, NCT00528489 and NCT01021059). Previous studies have shown that IL-15 promotes the survival, proliferation and Ig production of memory B cells41,42. In the current study, we examine human memory B cell IgG recall responses to H3N2 influenza virus in the presence of CpG2006 ODN activation with IL-15 co-stimulation in vitro. Our results demonstrate that stalk-reactive IgG antibodies induced by B cell exposure to H3 viruses in vitro, in the presence of CpG2006 ODN, are enhanced by IL-15 co-administration. In addition, IgG antibodies elicited by H3 viruses and/or IL-15 broadly bound to influenza HAs from both group 1 and group 2 influenza strains, which suggests potential use of CpG adjuvants and/or IL-15 agonists in influenza vaccination strategies.\n\n\nMethods\n\nThis study was approved by the Institutional Review Board at the University of Rochester Medical Center (RSRB protocol RSRB00066522). Subjects were recruited at the University of Rochester through local advertisement, and signed a written statement of informed consent prior to phlebotomy for the study. A total of 13 adults with an age range of 26 to 63 years (mean 43.7 years) were included in the study. Twelve study subjects (S1–S3, S5–S13) gave a history of being previously vaccinated with seasonal influenza vaccines, while one subject (S4) indicated that they had never received any influenza vaccine. Peripheral blood was obtained from all subjects as part of the study for B cell stimulation and analysis of baseline influenza-specific antibodies.\n\nThe levels of HA-reactive IgG were measured in plasma and in vitro stimulated B cell culture supernatants using the mPlex-Flu assay, as previously described43. The assay panels included whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA, as listed in Table 1. Briefly, 25µL of plasma dilution (1:5000) or undiluted culture supernatants were incubated with 25µL of a panel of beads coupled with HAs at room temperature for two hours on a rotary shaker (500 rpm) in the dark. Then 150µL of phycoerythrin (PE) conjugated goat anti-human IgG (Southern Biotech, Birmingham, Al) was added and incubated at room temperature for 2 hours on a rotary shaker (500 rpm) in the dark. After wells were washed twice with PBS (pH 7.2) containing 0.1% BSA (MP Biomedical, LLC, France) and 0.1% Brij-35 (Thermo Scientific, Waltham, MA), IgG levels were analyzed on Magpix Multiplex Reader (Luminex, Austin, TX). All samples were measured in duplicate.\n\nBPL inactivated virus used for in vitro stimulation only\n\n† Recombinant HA used in mPlex-Flu and BPL inactivated virus for in vitro stimulation\n\n‡GISAID accession number\n\nPrimary human B cells were isolated and activated with CpG2006 ODN, as previously described29,44. Cells were negatively enriched from peripheral blood by treatment with an EasySep Human B Cell Enrichment Kit (STEMCELL Technologies, Cambridge, MA), followed by magnetic separation according to the manufacturer’s instructions. B cells were resuspended in complete medium (RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin G, and 100µg/mL streptomycin) and were cultured (5x105/well, 1 mL/ well) with 6 µg/mL CpG2006 (Integrated DNA Technologies, San Diego, CA) alone or together with 10 µg/mL of BPL inactivated A/Victoria/361/2011 (A/Vic11, H3N2, IRR catalog No: FR-1041), A/Shanghai/1/2013 (A/SH13, H7N9, IRR catalog No: FR-1281), A/Hong Kong/33982/2009 (A/HK68, H9N2, IRR catalog No: FR-775) and pH1N1 viruses (H1N1, IRR catalog No: FR-187), or/and 50 ng/mL IL-15 (BD Pharmingen, San Diego, CA). Six days after incubation at 37°C 5% CO2, supernatant and B cells were collected for detection of anti-HA antibodies and ASCs, respectively.\n\nELISpot assay of memory B cell IgG secretion was performed as previously described25. Immobilon P membrane-based 96-well plates (Millipore, Billerica, MA) were coated overnight at 4°C with 10µg/mL H3N2 HA in phosphate-buffered saline (PBS) (40 µl/well). PBS only was added to the negative-control wells. Plates were blocked with complete PRMI 1640 medium. Cells were plated at a density of 106 per well in U-bottom plates and stimulated with CpG2006 ODN, six days after stimulation with A/Vic11 (H3N2, IRR catalog No: FR-1041). B cells were resuspended in complete medium containing either alkaline phosphatase-conjugated goat anti-human IgG (H-L) (KPL, Gaithersburg, MD) at 0.2µg/mL and incubated for 5h at 37°C in 5% CO2. The plates were washed, and then HA antibody secreting cell spots were developed with an alkaline phosphatase substrate kit (Vector Laboratories, Burlingame, CA). Spots were counted using a CTL ImmunoSpot plate reader and counting software (Cellular Technology Limited, Cleveland, OH).\n\nFor multiple comparison of H3N2-specific IgG between-group (CpG2006 ODN alone or together with H3N2 or H7N9 viruses), one-way ANOVA was performed. Correlations between H3-specific antibodies and stalk-reactive antibodies, or A/Vic11-specific antibodies between plasma and CpG2006 ODN ± memory B cells, were evaluated using the Pearson’s χ-squared test. Statistical analysis was performed using SPSS11.5 statistical software, as well as Prism 5 software. For all the statistical tests performed, p<0.05 was accepted as significant.\n\n\nResults\n\nTo evaluate the anti-influenza antibodies in plasma from 13 subjects and further infer influenza-specific memory B cells in their peripheral blood, we examined H3 HA-specific IgG levels using our mPlex-Flu assay. Six H3N2 strains, accommodating 45 years of antigenic drift of the swine origin influenza viruses (1968 to 2013), were selected to monitor the anti-H3 antibodies, including A/Hong Kong/1/1968 (A/HK68), A/Port Chalmers/1/1973 (A/PC73), A/Perth/16/2009 (A/Perth09), A/Victoria/361/2011(A/Vic11), A/Texas/50/2012 (A/Tex12) and A/Switzerland/9715293/2013 (A/Swi13). Antibodies against H3 strains were detected in all subjects. Of these, eight displayed high levels of H3-specific antibodies, while the other five had lower plasma baseline H3-reactive antibodies. Antibodies directed against A/Vic11 were high among all H3-specific IgG in 12 of the subjects. Only one subject displayed antibodies targeting the historical outbreak H3N2 strains, A/HK68 and A/PC73, which were higher than those against the more recent seasonal strains, A/Perth09, A/Vic11, A/Tex12 and A/Swi13 (Figure 1). The emergence of H3N2-specific antibodies indicates that all subjects likely had prior exposure to H3 influenza viral antigen.\n\nPlasma was obtained from 13 donors, 12 of which (S1–S3, S5–S13) had been previously vaccinated within the past 5 years with trivalent or quadrivalent seasonal influenza vaccines. Plasma baseline anti-influenza IgG was measured by mPlex-Flu assay. (A) Levels of IgG against distinct H3N2 strains. Each column depicts median fluorescence intensity (MFI), representing an individual donor. (B) Levels of H3 stalk-reactive IgG. Each symbol and line represents one donor. (C) IgG-binding to chimeric cH5/1 and cH5/3 proteins. This analysis was performed at a 1:5,000 dilution. Data can be found in Dataset 145.\n\nIn order to determine the degree of H3 stalk-reactive antibodies induced by inactivated A/Vic11 viruses, cH5/3, a soluble HA construction that contains the stalk of H3 virus and the head of H5 virus, was used in our mPlex-flu assay. This recombinant HA protein allows for the direct detection of stalk-reactive IgG antibodies in polyclonal sera or plasma. As shown in Figure 1B, H3 stalk-reactive IgG was detected in plasma (dilution of 1:5,000) from 11 donors, with 4 to 11-fold lower than H3 strain-specific antibodies. For 4 of them, MFI values were greater than 3,000. Since most donors had a history of receiving seasonal influenza vaccines, we also examined the H1N1 stalk-reactive antibodies. Consistent with H3 stalk results, H1 stalk-binding IgG was detected in all subjects (Figure 1C).\n\nTo evaluate memory B cell response to H3N2 viruses, purified B cells were stimulated with CpG2006 ODN and BPL inactivated A/Vic11 virus. Six days after stimulation, ASCs for H3 HA from A/Vic11 were detected in both CpG2006 ODN with and without H3 virus (CpG-H3) groups. As shown in Figure 2A, the number of ASCs was greater in CpG-H3 group than in CpG alone group. H3N2 strain-specific antibodies in supernatants were assessed by mPlex-Flu assay. B cells from all donors displayed rapid antibody responses to inactivated A/Vic11 viruses. Stimulation with CpG2006 ODN and H3 viruses resulted in a significant increase in antigen-specific IgG production, compared with H3, CpG and CpG with A/Shanghai/1/2013 (A/SH13) (CpG-H7) groups (Figure 2B). We also analyzed the relationship between the levels of A/Vic11-specific antibodies in plasma and IgG production by activated B cells. No correlation between antibody recall responses and plasma baseline IgG against A/Vic11 was detected (r=0.124, P=0.687) (Figure 2C).\n\nPurified B cells were obtained by negative selection and stimulated with CpG2006 ODN alone or together with A/Vic11 (H3) or A/SH13 (H7) control BPL inactivated virus for 6 days in vitro. (A) ASCs for H3 HA were examined by ELISpot assay. H3-binding antibodies in supernatants of activated B cells were monitored using beads bound with HA from H3N2 strains. (B) A/Vic11-specific IgG. Each symbol represents an individual donor. One-way AVONA was used to evaluate the difference among different groups ( P<0.05; ** P<0.01). Results were verified by ELISpot after stimulation with A/Vic11. The values represent the number of anti-HA IgG specific antibody-secreting cells in 1.25x105 stimulated B cells/donor. (C) Correlation between anti-A/Vic11 IgG levels in plasma and secretion of A/Vic11-specific IgG by activated B cells. (D) Antibodies binding to H3 clades. (E) Comparison of A/Vic11-specific IgG and A/Swi13-reactive IgG. Data from Dataset 246.\n\nWe next measured clade cross-reactive IgG induced by A/Vic11 viruses against selected recombinant HAs from seven heterovariant H3N2 strains spanning 45 years (1968–2013), A/HK68, A/PC73, A/Alabama/1/1981 (A/Ala81), A/Panama/1/2007/1999 (A/Pan99), A/Perth09, A/Tex12, and A/Swi13. Following stimulation with recent seasonal virus A/Vic11, activated B cells from all donors showed increased production of IgG targeting recent seasonal strains (A/Perth09, A/Vic11, A/Tex12 and A/Swi13), while increases in IgG against historical strains (A/HK68, A/PC73, A/Ala81 and A/Pan99) were detected in 11 donors (84.6%). One subject who had low baseline A/Vic11-specific antibodies showed much weaker antibody recall responses to A/Pan99, A/Perth09, A/Vic11, A/Tex12 and A/Swi13 strains than to the historical strains, A/HK68, A/PC73 and A/Ala81. In another subject, low levels of anti-A/HK68, A/PC73, A/Ala81 and A/Pan99, but high levels of anti-A/Perth09, A/Vic11, A/Tex12 and A/Swi13 were present after in vitro B cell stimulation (Figure 2D). Interestingly, antibodies against the most recent seasonal strain A/Swi13 were lower than those against A/Vic11 (Figure 2E).\n\nWe then tested the IgG induced by inactivated H3N2 viruses binding to recombinant HAs from influenza A strains, H1, H2, H5, H6, H7, H9, and B strains. For H1, six strains accommodating 89 years of antigenic drift of H1N1 influenza viruses from 1918 to 2009 were selected, including A/South Carolina/01/1918 (A/SC18), A/Puerto Rico/8/1934 (A/PR8), A/USSR/90/1977 (A/USSR77), A/Texas/36/1991 (A/Tex91), A/New Caledonia/20/1999 (A/NewCal99) and A/California/07/2009 (pdm A/Cali09). A/Japan/305/1957 (A/Jap57), A/Vietnam/1204/2004 (A/Viet04), A/Taiwan/2/2013 (A/TW13), A/TW13 and A/gf/HK99 represent H2, H5, H6 and H9 respectively, which are members of group 1. For group 2 influenza viruses, A/rhea/North Carolina/39482/1993 (A/rheaNC93), A/mallard/Netherlands/12/2000 (A/malNeth00) and A/SH13 represent three heterovariants of H7N9. B/Brisbane/60/2008 (B/Bris08), B/Mass/02/2012 (B/Mas12) and B/Phuket/2013 (B/Phu13) were selected for monitoring the cross reactivity to influenza B strains.\n\nAs shown in Figure 3A, antibodies binding to group 1 and B strains, induced by the group 2 subtype, H3 viruses, were detected in supernatants derived from 8 and 4 donors, respectively, with increases of median of 3.6 to 166.4-fold. IgG in supernatants derived from 8 donors displayed broad cross-reactivity to HAs from 6 different H1N1 strains. Anti-A/Jap57 IgG was detected in supernatants of activated B cells from 3 donors, while anti-A/Viet04, anti-A/TW13 and anti-A/gfHK99 IgG were detected in B cells from 2 donors. For group 2, increases in levels of IgG against H7N9 strains, A/rheaNC93, were detected in 4 donors. For influenza B strains, B cells from 4 donors showed increases in yield of cross-reactive antibodies. Increases in anti-B/Bris08 IgG were shown in 3 donors, while two donors demonstrated an increase in anti-B/Phu13. Increases in anti-B/Wis10 and anti-B/Mass12 were not detected. As shown in Figure 3B, the largest response was enhancement of anti-H3 B cell recall responses, and a broad, but lower, increase in responses to other more molecularly distant strains also occurred. Notably, significant increases in IgG binding to chimeric HAs containing the H3, H1, and H7 HA stalk segments, but not the H7 or H5 head segments, were observed, strongly suggesting the presence of broadly-cross-reactive stalk antibodies targeting the conserved stalk regions (Figure 3B).\n\nB cells were obtained by negative selection, and then stimulated with CpG2006 ODN alone or together with A/Vic11 for 6 days. Cross-reactive antibodies binding to H1, H2, H5, H6, H7 and B influenza subtypes in B cell culture were measured by mPlex-Flu assay43. (A) Fold change in cross-reactive antibodies. All values of IgG levels (MFI) were subtracted from those of medium before calculating fold change. Only those values of IgG induced by CpG2006 ODN plus A/Vic11 viruses, which were greater than 100, were selected to calculate fold change. Each symbol represents the median of fold change in levels of IgG induced by CpG2006 ODN with H3 to IgG stimulated by CpG2006 ODN alone. (B) CpG2006 ODN with H3 antigen induces a broad recall response to H3 influenza strains. Increases anti-HA IgG production to other non-H3 strains also occurred, but to a much lower extent. Data can be found in Dataset 347.\n\nH3 stalk-reactive antibodies were detected in activated B cells from 7 donors (53.8% of 13) after A/Vic11 stimulation (Figure 4A). IgG against historical outbreak strains (e.g. A/HK68, A/PC7), which have divergent head domains but conserved stalk domains with the recent seasonal H3N2 strains, was detected in most donors (12 of 13). Therefore, we analyzed the relationship between stalk-reactive and A/HK68 or A/Vic11 strain-specific antibodies. There was a strong correlation between cross-reactive stalk antibodies and A/HK68 strain-specific IgG production (r=0.945, P<0.001). Interestingly, anti-A/Vic11 displays a significant but weaker correlation with cH5/3 stalk antibodies (r=0.758, p=0.03) than those against A/HK68(Figure 4B). To further investigate the reactivity of cross-reactive stalk antibodies induced by influenza A subtype strains to H3 stalk, B cells from were stimulated with HA proteins from A/SH13 (H7; group 2) and pandemic A/California/07/2009 (pdm A/Cali09, H1; group 1) and A/Hong Kong/33982/2009 (A/HK09) (H9; group 1). Positive correlations between anti-cH5/3 and anti-HK68 IgG induced by A/SH13 (r=0.563, p=0.045) and pdm A/Cali09 (r=0.917, p=0.001) were detected (Figure 4C).\n\nB cells from healthy donors were stimulated with CpG2006 ODN alone or together with inactivated A/Vic11 (H3N2), A/SH13 (H7N9), A/Hong Kong/33982/2009 (A/HK09) (H9N2) or pdm A/Cali09 H1N1 viruses. The levels of IgG against H3 HA, H5 head and chimeric HA cH5/3 (H5 head and H3 stalk) are shown for individual subjects. (A) Nine of 13 subjects displayed increases in stalk-reactive IgG after A/Vic11 (H3) stimulation. (B, C) A correlation model assuming different coefficients for different anti-H3 strain-specific antibodies were fitted to evaluate the relationship between HA stalk-reactive and strain-specific IgG. Ordinate and abscissa units are mean fluorescence intensity (MFI). Data can be found in Dataset 448.\n\nTo assess if IL-15 has an influence on B cell recall responses to H3N2 viruses, we added IL-15 to cell cultures with CpG2006 ODN and BPL inactivated A/Vic11 influenza virus, or with CpG2006 ODN alone. The supernatants at day 6 were measured for IgG production against influenza A strains, including group 1 and group 2, B strains and chimeric HA proteins by mPlex-Flu assay. Fold change values were calculated as described in Figure 3. As shown in Figure 5A, cross-reactive antibody responses to the specific A/Vic11 viruses were enhanced in cell culture derived from all donors upon IL-15 stimulation.\n\n(A) B cells from healthy donors were co-stimulated with CpG2006 ODN, inactivated A/Vic11 viruses and IL-15. Strain-specific and HA stalk-reactive IgG in supernatants of activated B cells were detected after 6 days. (B) Fold change in cross-reactive antibodies. (C) Correlation between influenza specific antibodies and HA antigenic sequence. IL-15 increased the concentration of anti-HA reactive IgG, but does not alter the distribution of cross-strain specificity. Data can be found in Dataset 549.\n\nFirst we analyzed the influence of exogenous IL-15 on the in vitro B cell recall production of A/Vic11-specific IgG from all subjects. All subjects showed increased B cell secretion of A/Vic11 HA-specific antibodies after IL-15 treatment (median 13.1). Increases in cross-reactive antibodies binding to H3N2 heterovariant, A/HK68, A/PC73, A/Perth09, A/Tex12 and A/Swi13 were detected in all subjects, with median of 7.3, 9.4, 6.9, 8.2, 15, respectively. For influenza A subtype strains, IL-15 showed strong upregulation, with fold change in IgG against A/SC18 (median 16.6), A/PR8 (median 8.2), A/USSR77 (median 23), A/Tex91 (17.5), A/NewCal99 (20.3) and pdm A/Cali09 (14.3), A/Jap57 (42.6), A/Viet04 (28.3), A/TW13 (35.6), A/rheaNC93 (21.4) and A/gfHK99 (21.1). Ten donors displayed increases in IgG against B subtypes, B/Bris08 (median 44.6), B/Mass12 (17.8) and B/Phu13 (11.6) (Figure 5B). We next analyzed the relationship between strain-reactive IgG and HA stalk types, which revealed increases in stalk-reactive IgG against H1, H3, and H7 stalk regions that increased greatly with IL-15 + CpG2006 ODN stimulation in vitro. (Figure 5C).\n\nAlthough stalk-reactive antibodies induced by seasonal H3N2 viruses were detected in this study, these antibodies were lower than those against entire H3 HA. To assess whether H3 stalk-reactive antibodies can be enhanced by IL-15, we measured IgG binding to cH5/3. As shown in Figure 6A, anti-cH5/3 IgG increased after costimulation with CpG2006 ODN, H3 viruses and IL-15, compared with CpG-H3 and CpG groups. Since most influenza subtypes, either influenza A, including group 1 and group 2 subtypes, or influenza B strains, share conserved stalk epitopes, we also evaluated IgG against H7 and H1 stalk using cH5/1 (containing H5 head and H1 stalk) and cH4/7 (containing H4 head and H7 stalk). Anti-cH5/1(Figure 6B) and anti-cH4/7 (Figure 6C) IgG were higher in CpG-H3-IL-15 groups than in CpG-H3 and CpG alone groups. H7 and H1 stalk-reactive antibodies were upregulated along with H3 stalk-reactive antibodies.\n\nPurified B cells were costimulated with CpG2006 ODN, A/Victoria/361/2011 viruses and IL-15. Stalk-reactive IgG in supernatants was detected at day 6. Chimeric molecules cH5/3, cH4/7 and cH5/1 were used to measure antibodies against the H3, H7, and H1 stalks, respectively. Each symbol and line represents an individual donor. (A) H3 stalk-reactive antibodies. (B) H7 Stalk-reactive antibodies. (C) H1 stalk-reactive antibodies. Data can be found in Dataset 650.\n\n\nDiscussion\n\nThe majority of adults possess pre-existing IgG antibodies against influenza viruses from prior infection and/or vaccination51, primarily directed against the immunodominant globular head domain of the HAs. Antibodies against the conserved, immuno-subdominant HA stalk domain are generally detectable at very low levels, if at all, despite being broadly protective against multiple influenza strains and subtypes52. In this study, we analyzed strain-specific and H3 stalk-reactive antibodies in plasma from donors, and found high levels of anti-H3 specific IgG in all donors. Of these, 12 subjects had high IgG levels against the recent seasonal H3N2 strain A/Vic11, likely by vaccination or infection. One donor, who had never received a seasonal influenza vaccine, showed much lower IgG binding to A/Vic11, but higher anti-A/HK68 IgG levels. Interestingly, 11 of 13 subjects had detectable H3 stalk-reactive IgG at levels only 4–11 fold lower than H3 strain-specific IgG antibodies. The presence of pre-existing stalk-reactive antibodies suggests that stalk-specific memory B cells exist in memory B cell pool, a hypothesis supported by the results from the in vitro B cell stimulation ELISpot experiments.\n\nImmunological memory against influenza following immunization is the corner-stone for prophylactic vaccination programs53. Most adults possess a low (0.1–1.0% of total IgG memory B cells) but consistent base line of influenza virus-specific memory B cells25. Stalk-reactive memory B cells have generally been reported at very low frequencies, suggesting minimal effective protection16,20,54. Interestingly, broad cross-reactive antibodies have been detected in participants who received the pdm A/Cali09 vaccine24, which is distinct from the prior seasonal H1N1 strain. Competition of memory B cells responding to either common or rare antigens is hypothesized to regulate the appearance of cross-reactive IgG against influenza HA20,24. Repeat exposure to common influenza strains primarily boosts head-reactive responses and limits the expansion of B cells secreting broadly neutralizing antibodies. In contrast, repeated exposure to diverse influenza strains boosts antibodies against highly conserved influenza HA regions, such as the the stalk24 or a conserved Ca2+-binding region55 on the globular head.\n\nIn this study, we focused on enhancing the memory B cell recall response to H3N2 subtype influenza viruses following H3 stimulation in vitro in the presence of CpG2006 ODN ± IL-15. Class switched IgG antibodies against a panel of H3 strains spanning 45 years from 1968 to 2013 were analyzed. Increases in antibodies against recent seasonal strains were present in all subjects, while induction of IgG antibodies binding to historical H3 strains (prior to the birth year of the subjects) only occurred in 11 subjects. This is consistent with the prevailing hypothesis that antigenic drift leads to low vaccine efficacy5–7,10. Decreases in responses to Anti-A/Swi13 also explain the low vaccine efficacy in 2014–2015 influenza season. Moreover, we found that antibodies induced by H3 viruses, especially in the presence of IL-15, broadly bound to group 1 HAs, with moderate reactivity against group 2 and B influenza subtypes. These results are consistent with findings by other groups of cross-reactive IgG that react against both H1 and H3 influenza strains, primarily against epitopes on the HA stalk regions19,21.\n\nCpG2006 ODN stimulation has been shown to drive in vitro differentiation of both CD27− and CD27+ human B cells to the plasma cell phenotype29,44. Antibody production rates after CpG2006 ODN stimulation appear to be modulated by IL2Rγ signaling cytokines29. In vivo, mouse vaccine studies have noted that CpG adjuvanted influenza vaccination increases anti-HA IgG titers in young, but not older mice56. Our results suggest that the stimulation of B cells in vitro with inactivated H3 influenza in combination with CpG2006 ODN and IL-15 not only stimulate increased anti-HA IgG, but appears to increase levels of secreted cross-reactive IgG compared with CpG alone. IL-15 has been reported to overcome immundominance of antigens in CD8 T cell activation57. Further work will need to be done to determine if the addition of IL-15 to CpG adjuvanted influenza vaccines would boost protective anti-HA IgG production in vivo in older individuals.\n\nPrior characterization of broadly neutralizing IgG antibodies by other groups has demonstrated that cross-activity results from stalk-reactive antibodies26,58. Using the mPlex-Flu assay, we found that moderate to high levels of H3 stalk-reactive antibodies could be induced in vitro after CpG2006 ODN stimulation of memory B cells from 8 subjects (53.3%). These H3 stalk-reactive antibodies emerged along with IgG that bound the stalk regions of H1 and H7 subtypes, which share conserved epitopes with H3 viruses. Using a liner correlation model, we found that levels of antibodies binding to A/HK68 were positively related to H3 stalk-reactive antibodies, suggesting that clade cross-reactivity was likely due to the conserved epitopes in H3 stalk. This correlation also strongly existed between IgG antibodies binding to the H3 stalk and anti-A/HK68 antibodies responding to inactivated H7N9 viruses, suggesting that H3 stalk-specific memory B cells responded to H7N9 subtypes. Interestingly, stalk-specific IgG recall responses were not seen in memory B cells from the infected/un-vaccinated participant (S4), although they had detectable IgG antibodies against historical outbreak H3 strains after in vitro CpG2006 ODN + IL-15 stimulation.\n\nAlthough the production of stalk-reactive antibodies indicated activation of stalk-specific memory B cells, we found both strain cross-reactive and stalk-reactive IgG antibodies present at much lower levels than H3 strain-specific anti-HA IgG antibodies. Given the desirability of inducing broadly cross-reactive anti-HA stalk-reactive IgG antibodies, vaccination strategies to achieve this goal need to be developed. In this study, we evaluated the effect of supplemental IL-15 on B cell recall responses to inactivated A/Vic11 viruses. We demonstrated that the B cell recall responses to H3 viruses were enhanced by CpG2006 ODN + IL-15, with increases of 6.9 to 15-fold in IgG production. Broadly cross-reactive IgG antibodies were observed to bind to group 1, group 2 and B strain influenza HA subtypes following CpG2006 ODN + IL-15 stimulation, with median of increase of 22.3-fold for group 1, 21.4-fold for both group 2 and 17.6-fold for B subtypes compared to CpG2006 ODN stimulation alone. This demonstrated IL-15 greatly augmented recall secretion of broadly cross-reactive anti-influenza HA IgG antibodies. Not surprisingly, there was a negative correlation between antigenic sequence dissimilarity of the stimulating influenza strain and recall IgG antibody levels.\n\n\nConclusions\n\nIn conclusion, broadly cross-reactive anti-influenza stalk-binding IgG antibodies exist in individuals exposed to influenza strains. Seasonal H3N2 virus exposure, through vaccination or infection, can induce memory B cells that bind to the conserved stalk region of HAs. In vitro recall responses to these stalk-reactive antibodies can be enhanced by IL-15. These results suggest the potential for IL-15 augmentation of adjuvant to overcome immunodominance of influenza HA head region epitopes as a potential vaccine boosting strategy to increase levels of broadly cross-reactive anti-influenza HA IgG antibodies.\n\n\nData availability\n\nFigshare: Dataset 1: Anti-H3 stalk reactive antibodies in human plasma. https://doi.org/10.6084/m9.figshare.5481565.v245\n\nFigshare: Dataset 2: Secretion of H3 clade cross-reactive antibodies by B cells stimulated with inactivated A/Vic11. https://doi.org/10.6084/m9.figshare.5498080.v146\n\nFigshare: Dataset 3: Influenza viruses induce cross-reactive antibody responses in vitro. https://doi.org/10.6084/m9.figshare.5498071.v247\n\nFigshare: Dataset 4: Induction of HA stalk-reactive antibodies by H3 viruses. https://doi.org/10.6084/m9.figshare.5498116.v148\n\nFigshare: Dataset 5: IL-15 increases cross-reactive antibody responses to H3N2 viruses. https://doi.org/10.6084/m9.figshare.5498152.v149\n\nFigshare: Dataset 6: Stalk-reactive antibody responses to H3 viruses enhanced by Il-15. https://doi.org/10.6084/m9.figshare.5498197.v150\n\nAll data are available under the terms of the Creative Commons Attribution 4.0 International license (CC BY 4.0).", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis research was supported by the China Scholarship Council and the Zunyi Medical University Visiting Scholar Grant (201408525075; JH), the National Institutes of Health, National Institute of Allergy, Immunology and Infectious Diseases (grants HHSN272201000055C, HHSN272201400008C, AI098112, AI069351, AI109946; JW, SH, MSZ, JG), and the University of Rochester Medical Center, Clinical and Translational Science Institute (CTSI) (grant UL1TR00042; JW, MSZ).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgements\n\nThe authors would like to thank Sheldon Perry for technical assistance.\n\n\nReferences\n\nWorld Health Organization: WHO Weekly Epidemiological Record. 2016; 91(52): 601–624. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRamanathan MP, Kutzler MA, Kuo YC, et al.: Coimmunization with an optimized IL15 plasmid adjuvant enhances humoral immunity via stimulating B cells induced by genetically engineered DNA vaccines expressing consensus JEV and WNV E DIII. Vaccine. 2009; 27(32): 4370–80. PubMed Abstract \| Publisher Full Text\n\nLim KL, Jazayeri SD, Yeap SK, et al.: Antibody and T cell responses induced in chickens immunized with avian influenza virus N1 and NP DNA vaccine with chicken IL-15 and IL-18. Res Vet Sci. 2013; 95(3): 1224–34. PubMed Abstract \| Publisher Full Text\n\nGill N, Paltser G, Ashkar AA: Interleukin-15 expression affects homeostasis and function of B cells through NK cell-derived interferon-gamma. Cell Immunol. 2009; 258(1): 59–64. 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Data Source\n\nHuang J, Zand M, Wang J, et al.: Dataset 5: IL-15 increases cross-reactive antibody responses to H3N2 viruses. figshare. Retrieved: 13:45, Nov 07, 2017 (GMT), 2017. Data Source\n\nHuang J, Hilchey SP, Wang J, et al.: Dataset 6: Stalk-reactive antibody responses to H3 viruses enhanced by Il-15. figshare. Retrieved: 13:45, Nov 07, 2017 (GMT), 2017. Data Source\n\nSasaki S, Jaimes MC, Holmes TH, et al.: Comparison of the influenza virus-specific effector and memory B-cell responses to immunization of children and adults with live attenuated or inactivated influenza virus vaccines. J Virol. 2007; 81(1): 215–28. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMoody MA, Zhang R, Walter EB, et al.: H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination. PLoS One. 2011; 6(10): e25797. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nMelchionda F, Fry TJ, Milliron MJ, et al.: Adjuvant IL-7 or IL-15 overcomes immunodominance and improves survival of the CD8+ memory cell pool. J Clin Invest. 2005; 115(5): 1177–87. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTan GS, Lee PS, Hoffman RM, et al.: Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin. J Virol. 2014; 88(23): 13580–92. PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "28740", "date": "08 Jan 2018", "name": "Qibo Zhang", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors studied anti-influenza HA IgG antibody levels using a multiplex assay in adults who exposed to H3 viruses previously and/or received influenza vaccination and demonstrated that stalk-reactive antibody recall responses induced by seasonal H3 viruses and CpG ODN can be enhanced by IL-15. The manuscript is generally well written and clearly presented. It may be helpful that authors could add a bit more discussion as to how IL-15 with/without CpG activate the B cell recall response leading to the anti-stalk antibody production.\nMinor point: page 9, top right \"B cells from ?? were stimulated with HA proteins from A/SH13\"\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "29326", "date": "02 Feb 2018", "name": "Raghavan Varadarajan", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nReviewer comments: In the current study, authors examined the human memory B cell IgG recall responses to H3N2 influenza virus in the presence of CpG2006 ODN activation with IL-15 co-stimulation in vitro. They demonstrated that stalk reactive IgG antibodies induced by B cell exposure to H3 viruses in vitro, in the presence of CpG2006 ODN, are enhanced by IL-15 co-administration. In addition, IgG antibodies elicited by H3 viruses and/or IL-15 broadly bound to influenza HAs from both group 1 and group 2 influenza strains, which suggests potential use of CpG adjuvants and/or IL-15 agonists in influenza vaccination strategies. The use of IL-15 to enhance responses to immunization has been studied previously (see for example, PMID:24706798 which the authors could cite). However in that study, it was T-cell rather than antibody responses that were found to be protective. In this context, it would be important in a future study to examine whether the induced, cross-reactive antibodies show any protective activity in an appropriate assay. Overall, the manuscript is clearly written and interesting to read. However, the authors could consider the following additional points to improve clarity.\nPlease include list of abbreviations used in the manuscript.\n\nPage No. 4. What are the concentration of whole HA or the head segments of influenza group 1, group 2, B strain and chimeric HA used for the mplex-Flu assay? What is the concentration of PE conjugated goat anti-human IgG (mplex-Flu assay) used in the current study?\n\nPage No. 5. On what basis was the concentration of IL-15 (50 ng/mL) fixed for the study? Did the authors check effects of different concentrations? If yes, what was the effect? (It is important to screen the IL-15 concentration because, in a previous NHP study antibody responses were sensitive to IL-15 dose with a slight increase at lower doses, but a decrease at higher doses (Yin et al., High dose of plasmid IL-15 inhibits immune responses in an influenza non-human primates immunogenicity model. Virology; 393(1), 2009, 49-55.).\n\nIn the result section, authors mention “As shown in Figure 1B, H3 stalk-reactive IgG was detected in plasma (dilution of 1:5,000) from 11 donors, with 4 to 11-fold lower than H3 strain-specific antibodies. For 4 of them, MFI values were greater than 3,000”. However, the maximum MFI values given in data set is 1250. Also there is no caption for Figure 1C.a\n\nPage No. 9. H2N2 should be changed to H3N2.\n\nUse A/Vic11 abbreviation consistently throughout the manuscript.\n\nReferences should be uniform.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nNot applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2015
https://f1000research.com/articles/6-465/v1	12 Apr 17	{ "type": "Software Tool Article", "title": "Introducing the Brassica Information Portal: Towards integrating genotypic and phenotypic Brassica crop data", "authors": [ "Annemarie H. Eckes", "Tomasz Gubała", "Piotr Nowakowski", "Tomasz Szymczyszyn", "Rachel Wells", "Judith A. Irwin", "Carlos Horro", "John M. Hancock", "Graham King", "Sarah C. Dyer", "Wiktor Jurkowski", "Annemarie H. Eckes", "Tomasz Gubała", "Piotr Nowakowski", "Tomasz Szymczyszyn", "Rachel Wells", "Judith A. Irwin", "Carlos Horro", "John M. Hancock", "Graham King", "Sarah C. Dyer" ], "abstract": "The Brassica Information Portal (BIP) is a centralised repository for Brassica phenotypic data. Trait data associated with Brassica research and breeding experiments conducted on Brassica crops, used as vegetables, for livestock fodder and biofuels, is hosted on the site, together with information on the experimental plant materials used, as well as trial design. BIP is an open access and open source project, built on the schema of CropStoreDB, and as such can provide trait data management strategies for any crop data. A new user interface and programmatic submission/retrieval system helps to simplify data access for scientists and breeders. BIP opens up the opportunity to apply big data analyses to data generated by the Brassica Research Community. Here, we present a short description of the current status of the repository.", "keywords": [ "Brassica", "phenotypic trait data", "data integration", "FAIR data", "phenotyping API", "crop ontology", "genotype-phenotype association", "enhanced breeding" ], "content": "Introduction\n\nBrassica is a diverse genus that includes a number of species cultivated as important crops, providing edible roots, leaves, stems, buds, flowers and seed1. Due to their wide adaptation and ability to thrive under varying agroclimatic conditions2, Brassica crops are grown all over the world for food, animal fodder and industrial applications.\n\nGiven the importance of Brassica species worldwide, integrated approaches to research and plant breeding for crop improvement are required to address future challenges, such as satisfying increased demand for higher quality or producing reliable yields in a more variable environment with sustainable, lower input cultivation systems. Researchers have quantified many aspects of Brassica phenotypic diversity, including the content of industrially important compounds, yield in response to fertiliser input or degree of resilience to fungal pathogens and pests, such as flea beetles, to aid in crop improvement. The ability to host such data generated from these experiments in an open and accessible database promotes the comparison of trait measurements and calculation of genotype-phenotype (G X E) associations across multiple studies in meta-analyses, potentially identifying underlying common mechanisms or previously unrecorded correlations. This is especially important for plant breeders and researchers involved in pre-breeding, leading to generation of new crop varieties. For example, meta-analysis across different populations or environments can help confirm the location of key areas of the genome identified from multiple studies or assist and help avoid potential trait trade-offs of beneficial or deleterious alleles due to linkage drag. The ability to associate phenotypic data with genomic regions, DNA markers, and genome and transcribed sequence provides the potential to expedite the integration of favorable alleles via marker-assisted or genomic selection. However, few plant phenotype databases exist, despite the importance of phenotypic data to render meaning to sequence data.\n\nMaintenance of easily accessible and reusable plant phenotypic data is challenging because, in contrast to standardised approaches used in sequence analysis, traits can be measured with a variety of protocols whose methods are not captured automatically, and there is mostly no consensus on methodologies to record any given trait. Moreover, there is a lack of controlled vocabularies to describe phenotyping metadata adequately. One possible reason is that, as opposed to publishing sequencing data in databases widely used by the research community, such as ArrayExpress3, GEO (Gene Expression Omnibus)4, ENA (European Nucleotide Archive)5 and SRA (Sequence Read Archive)6, it is not a requirement to submit phenotyping data to a public repository to accompany peer-reviewed publications. There is, to date, no single plant phenotypic database recommended by Nature’s Scientific Data. Compared to sequence data, the formal description of trait and associated meta-data tends to be more complex and crop or species specific.\n\nData re-use and re-analysis can lead to new knowledge and enhancement in understanding. For example, readily available datasets retrieved from a database can serve 1) as evidence for new claims, 2) to explore existing knowledge and identify gaps, 3) to bridge between research domains or 4) to help with selecting research directions. Additionally, databases can serve as tools for collaboration across countries, fields or areas of expertise and link between research and industry.\n\nExisting Brassica-related databases and analysis tools mostly focus on Brassica genomic information. For example, BRAD7 hosts data on Brassica genomes and PlabiDB8 is a platform that, among others, hosts genomic data from Brassica napus. GnpIS is a complex system that besides sequence and genomic information aims to share transcriptome and phenotype data but at present these data is not publicly available. While these databases are useful in their own context, they do not focus on setting or demonstrate the value of community-wide phenotypic data standards, and therefore do not encourage universal trait sharing and data re-use.\n\nResources, such as TAIR (arabidopsis.org)9, or Araport (araport.org)10 and the 1001genomes project11, provide data storage and analysis tools for the Arabidopsis Scientific community. Due to the close evolutionary and taxonomic relatedness of the model plant Arabidopsis in the Brassicaceae, Arabidopsis-related databases or platforms for genotype and phenotype information provide valuable insights into the underlying Brassica biology, and potential insights into the crop analysis tools and resources of the future. However, current Arabidopsis resources are not directly applicable to recording genotype-phenotype specific traits of Brassica crops.\n\nCropStoreDB12, the first systematic Brassica phenotype database, was launched as part of InterStoreDB steming from previous efforts such as Integrated Marker System for Oilseed Rape Breeding13, and BrassicaDB14,15. CropStore hosts Brassica trait data, connecting it with relevant sequences in SeqStore for genotype and phenotype data integration.\n\nThe Brassica Information Portal builds on the CropStoreDB schema, and provides the technologies to enable efficient storage, classification and management of phenotyping data. Its unique features are the universally applicable Brassica trait Ontology, a Digital Object Identifier (DOI) for each dataset, login via ORCiD (https://orcid.org/), cross-references to sequence information and primary data, user-friendly wizard-based submissions, and Application Programming Interface (API) based data submission and retrieval. In addition, submitted data can be kept private for a user-defined period of time. Extensions for visual data exploration and tools for associative analysis are intended in the future releases, to provide a desired set of features for the Brassica community.\n\n\nMethods\n\nTo address the challenges facing a phenotypic trait database, the BIP has implemented rigorous use of ontologies and standards. One element is a dictionary of Brassica-specific traits created according to CropOntology16 for that purpose. The CropOntology phenotype annotation model is based on the combination of “trait method and scale”, as it is grounded in breeder’s datasetformats16. This makes it especially suitable for describing pre-breeding material traits hosted in the portal. The level of detail in the trait definition makes it possible to select meaningful traits for analysis. Hence, the CropOntology encourages a way of defining a trait to facilitate meta-analysis, integrated data analysis and data discovery, some of the main aims of the repository besides data storage.\n\nFurther incorporation of ontology includes plant anatomy terms from Plant Ontology17 and taxonomical classification from Gramene’s Taxonomy Ontology18. Cultivar names are curated, and submissions are checked against this curated list.\n\nInitial minimal requirements for trial, population and trait description have been identified and are made compulsory during submission, to associate meaningful experimental meta-data with the trait scores. BIP development is benefiting from discussions on standard minimal requirements for plant phenotyping experiment (MIAPPE) data currently ongoing as part of the Excelerate project run by ELIXIR consortium. MIAPPE functions as a checklist of attributes that can be used to describe each experimental unit19,20, therefore by representation of the most useful information captured by researchers it allows comprehensive data sharing and reuse. A similar approach has been applied previously for other data types, including gene expression microarrays (MIAME), sequencing (GSC), metabolomics (MSI), and proteomics (MIAPE).\n\nIn BIP, we are focusing on information related to recording Project, Trial, Sample, Location, Treatment and Trial Environment. Our contribution includes the definition of MIAPPE fields and proposal of ontologies to ensure there is standardised content to describe Brassica phenotyping experiments mapped to these requirements. With regard to file types, the choice of standard input and output file is the .csv format, as it is readable by most software tools and humans alike. Further, the API allows for machine readable .json output format.\n\nThe content of the database includes population data with member lines and diversity sets with lines representing cultivars or other ex-situ genetic resource material. Linked to this hierarchical relationship of cultivars and lines are trait scores with associated experimental and organisational metadata. As this repository has inherited its structure and content from CropStoreDB, it also hosts historical QTL and linkage map data linked to experimental segregating populations and trait data. For obvious reasons we do not intend to cater directly for sequencing data, yet we continue to host genotyping data generated through modern DNA, RNA sequencing or genotyping arrays. The BIP currently hosts data from numerous pre-breeding programmes that include 22 plant trials, with 139,000 trait scores from 188 different varieties and 26,100 plant lines.\n\nWhile the repository lays the foundation for more extensive integration of genotype and phenotype information and tools, it is already integrated with TGAC Browser21 to allow cross-linking of single nucleotide polymorphisms (SNPs) of interest to lines/cultivars in the BIP that may carry the corresponding SNP.\n\nThere are three main building blocks that constitute the BIP. The first element is the relational database, which stores and manages all BIP data. It uses PostgreSQL RDBMS technology. BIP’s initial data and the database schema were obtained from the CropStoreDB system and then underwent several transformations, including translation from the original MySQL format to the target PostgreSQL format, i.e. introduction of formal foreign keys and presence constraints, and a series of data curation procedures to ensure referential integrity. The BIP database is the core of the system, enacting all data management operations, issued both through the web interface and the REST API. The decision to transfer data to PostgreSQL was taken as it delivers more advanced, modern capabilities of data management than MySQL, while still being an open source and a free-of-charge solution. The overview of the database schema is presented in Figure 1.\n\nSome “operational” tables, which are used only internally by the system (e.g. for access rights), were omitted.\n\nThe second element is built on top of the database and is a modern web application. Its purpose is threefold: (i) it delivers advanced data browsing interfaces, which allow for data ordering, filtering and export to CSV documents (available to all users without registration; see Figure 2) (ii) it implements data submission wizards, explained in detail in the section below (for registered users only) (iii) and it gives the possibility of managing data through the API, which supports reading, loading, querying, searching, creating and publishing data.\n\nThe web application was created using the Ruby on Rails framework. All web requests are channeled through the Nginx web server, which in turn routes them to a web application logic server (a so-called reverse proxy setup). All traffic is secured with an encrypted HTTPS protocol. User registration is implemented using the third party authentication solution (orcid.org).\n\nThe third constituent element of the BIP is the data indexing and full-text search engine. It analyzes all records in every table of the database, including all new data being submitted by users, and creates a search index. The BIP web search functionality (present on the front page of the website; see Figure 2) and its API search capability (available via programmatic access to registered users) is integrated with the search engine in order to deliver the arbitrary term search. For every submitted search term, the engine first looks the term up in the index, and if found, responds with a set of data-table records that include this term. This element of the BIP is created using Elasticsearch technology.\n\nUsers initiate actions with either their web browsers or API clients (blue elements). All requests go through respective access control layers (these ensure e.g. that a given set of data is accessible to the user), before they contact the data management layer to retrieve (or commit) data from (to) the database. Access mode is read/write (solid arrows). Additionally the search engine could be required to search through gathered data (dashed arrows) - it’s a read-only access mode. Finally, the data layer informs the search engine about any new data to be indexed (the dotted arrow).\n\nAll three elements are presented in Figure 3, together with respective data transfer channels between them. Deployment-wise, the database system, the web application logic server, and the indexing and search engine all reside within a single system. If, in the future, the scale of the data managed by the BIP becomes too high for current machine capabilities, it is possible to decouple these elements in order to distribute the load on several physical servers.\n\nBIP operates at bip.earlham.ac.uk/ and is publicly accessible at this address, as a web resource. However, it is possible to use BIP open source to deploy one’s own BIP instance. In such a case, BIP will require a server, either physical or virtual, with at least two contemporary CPU cores, and at least four gigabytes of operational memory. Due to its internal architecture, described in the previous section, two processor cores are required to achieve a smooth user experience along with simultaneous maintenance of fast database and indexing engine responses. Since the DB and the web application itself makes a heavy use of disk caching mechanisms to provide faster response to user queries, it is also beneficial, though not absolutely required, to equip the server with fast hard drives. The storage space needed depends on the amount of data one plans to deposit in the instance of BIP, but the application itself has no high requirements in this respect, and 6 GB of storage space should be sufficient for both the operating system and all the components of BIP. Any up-to-date Linux operating system will be adequate to host BIP.\n\n\nUse of the BIP\n\nSeveral tables hosting interlinked population, trial, trait score, linkage map, QTL and marker assay data are accessible via the web-interface, either from a universal search, or by browsing the database for specific tables (Figure 2).\n\nAccessing related data is facilitated by following the “Related” button in case of the Populations table, or by clicking onto a cross-linked field within the table (these appear green; Figure 4A). On top of each table, a search bar can query for specific components within the table (Figure 4A). Organisational and provenance metadata are made visible by clicking the blue information button for each row (Figure 4B). Each column can be sorted, resulting in global reordering across the whole table (Figure 4B, red arrow). The visible content of the tables can be downloaded as a .csv file, by clicking on the respective button on top of the table (Figure 4B).\n\nA) refined querying and access to related data B) reordering of results, function for bulk download of data with accompanying meta-data, display of the meta-data.\n\nExperimental data is generally recorded in spreadsheet format. Therefore, both the web form wizard provided within the tool, and the available Ruby script client, use spreadsheet formats as templates to assist the user in uploading the data. Data are submitted in two separate logical batches. First, information about the experimental plant population needs to be submitted. Second, trait data from Trials performed on these plants are submitted during Trait Scoring Submission. This enables the submission of multiple trials per experimental Plant Population.\n\nA wizard-based submission of Population and Trial Data data guides the user through all compulsory and optional field contents that can be submitted to the database. During Population Submission (Figure 5A), a four-step process asks the user to enter population related information, including the submission of the actual lines and associated metadata in a .csv template designed in step 3. The purpose of the template is to make it easier to appropriately format large data sets and avoids spelling mistakes in the uploaded file’s header. The standard template used during Population Submission is shown in Supplementary Figure 1B. Plant variety names are checked for their existence and correct spelling against the database. The final step is the submission of data provenance information. Compulsory metadata for Population Submissions is listed in Supplementary Table 1.\n\nDuring Trial Submission (Figure 5B), the first steps involve entering trial related information, before the traits studied in the trial are selected or defined in step 2. To avoid duplicated traits, the interface suggests traits based on an elastic search using two or more letters typed by the user. If traits are unavailable, the user can define and add them manually (the Plant and Trait Ontology vocabulary is provided to assist the user in this task). A high quality of trait definition and trait measurement-interoperability between experiments is ensured by interposing a human curator between submitter and database.\n\nFor the submission of trait measurements, the user designs a .csv template in step 3 (see Supplementary Figure 1A for an example template), and then submits the completed .csv file in step 4. Depending on the type of data submitted (raw vs. analysed data), the template can differ in the fields to be filled out. Compulsory metadata for Trial Submissions is listed in Supplementary Table 1. Finally, the user can upload an image presenting the experimental layout (for instance, the greenhouse plant pot setup on benches) in the case of raw data submission, and fill out provenance metadata in step 6. Whether data are submitted using the wizard or the API, the same metadata is compulsory.\n\nProgrammatic submission is executed using POST requests through the BIP API. The query exposes the part of the database data the user wants to submit data to. Content to be submitted needs to be assigned a corresponding field in the database and is presented as files in .json format. Programmatic extraction is also API based. GET requests placed to the database can be more comprehensive or specific than the interface-based strategy. These are then output in .json format, ready to feed into analytic workflows run in, for example, python. Full API documentation is available on the portal's website, including all table fields beyond those that are compulsory.\n\nA Ruby submission client facilitates the automated submission of new experimental populations and trial data. It parses an input .csv file searching for the elements (input columns) to be submitted. These input columns need previously to have been mapped against the database fields by the registered user. For each line in the .csv, new entries are created in the database and this content is then submitted to the database using the BIP API. Using the Ruby client tool is not required - it is provided only as a convenient alternative to users who prefer scripting approach to data submission and analysis.\n\nThe registered user is entitled to upload data directly to the database, which may then become public within seconds of submission. However, to ensure confidentiality of data for an ongoing project, at user’s discretion, a submission embargo can keep data private for a defined period. Once public and older than the revocation period, datasets could be assigned (on user’s demand) with individual DOIs, which act as a reference in publications.\n\nData download can be performed via the web interface and the API. Using the interface, the user can download any visible table, by clicking the “Export to CSV” button on top of the tables. This downloads the complete table in .csv format (Figure 4b). Via the API, the user can use GET requests to interact with the database tables directly. This sometimes enables the user to retrieve more information than is displayed at the interface. The queries can be crafted according to the user’s needs for data. Example Ruby clients are available on Github for modification and retrieval of more complex data.\n\n\nConclusions\n\nThe Brassica Information Portal (BIP) has been designed as an open-access, open-source, phenotype data storage resource and will facilitate addition of other features in the future. The general motivation for the BIP was to develop technologies enabling efficient use of phenotyping data. The creation of the database and its standards lays the foundation for Brassica genotype-phenotype studies drawing phenotypic data from this repository.\n\nAvailability of a repository for integrative phenotype data should encourage the Brassica research community to store trait data in an open access repository, in a similar manner as for other data types, such as sequence, metabolomic and protein structure data, and share it with the research community. Ultimately, the Portal will facilitate understanding of Brassica diversity and trait variation for future research, breeding programmes and crop improvement.\n\nWith instant submission to the repository, it remains the primary responsibility of the submitter to guarantee the quality of the data. However, cross-linking with existing ontologies and spelling control holds errors in check prior to submission. Further, compulsory fields during submission ensure metadata minimal requirements are met for meaningful data interpretation in the future. Furthermore, each submission is granted a one week revocation period. During that time, the submitting author is asked to perform all important data quality checks, and in case any discrepancies or inconsistencies are found, the author is able to revoke the publication, repair the submitted dataset, and publish it again. After a week has elapsed since the submission, the data are made read-only, and it can no longer be altered or removed. Moreover, it can then be accessed and quoted by other users as long as the embargo is lifted. The author may request a DOI number assigned to the submission, so it is possible to cite it in publications.\n\nFuture plans include the support of submission of imaging data to CyVerse, allowing BIP to serve as a single entry-point for multiple data types relevant to crop improvement studies. At the same time, visualisation tools will be developed to call and display the CyVerse-hosted images from within the BIP interface. This will include 1) images of single plant parts underlying definitions of specific traits; 2) time lapse images of single plants; and 3) time lapse images of field trials generated by high-throughput techniques, e.g. drone flights. Statistical analysis of phenotypic data will be performed with incorporated of R/Shiny applications run directly on the BIP server. For example, association studies could be performed with uploaded genotyping data matching selected plant lines with corresponding trait measurements available in the BIP. More computationally costly operations will be supported through development and incorporation of analytical workflows executed externally, e.g. using CyVerse infrastructure. Additionally, linkage markers recorded in the database will be cross-linked with their corresponding sequences within EnsemblPlants22. This will make it possible to visualise the location of the markers in context with the genome sequence and genes in close proximity.\n\nFinally, BIP API will be further refined by implementing additional content checks, e.g. by cross-linking to Global and European cultivar registers (Plant variety database - European Commission, Community Plant Variety Office, CPVO Organisation for Economic Co-operation and Development (OECD) Variety List Query) and to comply with ELIXIR standards (currently under development) to facilitate common data exchange and communication with other bioinformatics resources.\n\n\nSummary\n\nWe have introduced the Brassica Information Portal, a web repository for Brassica phenotype data. This article describes the current state of development and future plans. We describe the back end database schema and the front end web-interface. Also, we describe programmatic and wizard-based submission of Population and Trial data together with fields compulsory for submission to the repository. It was originally intended that this repository meet the needs of the Brassica Research Community in the UK for relevant experimental data. However, it is apparent from discussions within the Multinational Brassica Genome Project that it can now fulfill a broader community infrastructure requirements. We therefore hope that BIP encourages data sharing and re-use by helping to integrate and harmonise datasets generated worldwide.\n\n\nSoftware availability\n\nBrassica Information Portal can be accessed from: bip.earlham.ac.uk\n\nLatest source code: https://github.com/TGAC/brassica\n\nArchived source code as at time of publication: https://doi.org/10.5281/zenodo.46605023\n\nLicense: GNU 3.0", "appendix": "Author contributions\n\n\n\nWJ and SCD conceived and led the project; WJ, SCD, AHE, TG and PN designed the BIP. TG, TS and PN developed the BIP code base and implemented the bip.earlham.ac.uk instance. AHE prepared the first draft and TG, WJ, SCD contributed to preparation of the manuscript. AHE and RW carried out the data curation. AHE, SCD and WJ coordinated the development. CH and JMH mapped the BIP data model to MIAPPE and BrAPI requirements. RW, JAI and GK helped designing layout and BIP functionality and provided further user feedback. GK helped adopting CropStoreDB schema in BIP. All the authors were involved in revising the draft manuscript and have agreed to the final content.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThis work was supported by UK Biotechnology and Biological Sciences Research Council as part of the BBSRC Renewable Industrial Products from Rapeseed (RIPR) Programme (BB/L002124/1) and was strategically funded by the BBSRC, Institute Strategic Programme Grant (BB/J004669/1) at the Earlham Institute (formerly The Genome Analysis Centre, Norwich). CH is supported by ELIXIR-EXCELERATE, funded by the European Commission within the Research Infrastructures programme of Horizon 2020 (grant agreement number 676559).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nAcknowledgments\n\nWe thank Ian Bancroft (University of York, UK) and other members of RIPR consortium and UK Brassica community for support and feedback throughout the duration of this project. We are grateful to Thomas Alcock (University of Nottingham, UK) for battlefield tests of BIP API and data submission wizard. We would also like to show our gratitude to Paweł Krajewski and Hanna Ćwiek-Kupczyńska (Institute of Plant Genetics, Polish Academy of Sciences, Poland) for discussions on phenotyping data standards including Minimum Information About a Plant Phenotyping Experiment (MIAPPE); and to Manuel Corpas for introducing us to the ELIXIR programme, immensely important for future phases of BIP development.\n\nThis development was supported in part by the Scientific Computing Group at the Earlham Institute and the Computing infrastructure for Science (CiS) group at the NBI through help in configuring and hosting the BIP, and with access to the HPC resources available through Earlham Institute’s National Capability in Genomics.\n\n\nSupplementary material\n\nSupplementary Figure 1: Examples of .csv templates generated during (A) Trait Scoring Submission and (B) Population Submission. The latter can change substantially depending on the type of data submitted. In this case raw data is being submitted as the template asks for information on the trial design.\n\nClick here to access the data.\n\nSupplementary Table 1: List of fields that are compulsory during the submission of Populations and Trial (Trait Scoring) data.\n\nClick here to access the data.\n\n\nReferences\n\nRakow G: Species Origin and Economic Importance of Brassica. Springer Berlin Heidelberg, Berlin, Heidelberg. 2004; 54: 3–11. Publisher Full Text\n\nLabana KS, Gupta ML: Importance and Origin. In Breeding Oilseed Brassicas. Springer Berlin Heidelberg, Berlin, Heidelberg. 1993; 19: 1–7. Publisher Full Text\n\nBrazma A, Parkinson H, Sarkans U, et al.: ArrayExpress--a public repository for microarray gene expression data at the EBI. Nucleic Acids Res. 2003; 31(1): 68–71. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nEdgar R, Domrachev M, Lash AE: Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHuala E, Dickerman AW, Garcia-Hernandez M, et al.: The Arabidopsis Information Resource (TAIR): a comprehensive database and web-based information retrieval, analysis, and visualization system for a model plant. Nucleic Acids Res. 2001; 29(1): 102–105. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrishnakumar V, Hanlon MR, Contrino S, et al.: Araport: the Arabidopsis information portal. Nucleic Acids Res. 2015; 43(Database issue): D1003–9. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe 1001 Genomes Consortium: 1,135 Genomes Reveal the Global Pattern of Polymorphism in Arabidopsis thaliana. Cell. 2016; 166(2): 481–491. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLove CG, Andongabo AE, Wang J, et al.: InterStoreDB: a generic integration resource for genetic and genomic data. J Integr Plant Biol. 2012; 54(5): 345–355. PubMed Abstract \| Publisher Full Text\n\nBancroft L, Barnes S, Li JY, et al.: Establishment of an Integrated Marker System for Oilseed Rape Breeding (IMSORB). 2006. Publisher Full Text\n\nTrick M, et al.: Brassicadb is a database of genetic and molecular information derived from key brassica species. 2003. Reference Source\n\nQiu D, Morgan C, Shi J, et al.: A comparative linkage map of oilseed rape and its use for QTL analysis of seed oil and erucic acid content. Theor Appl Genet. 114(1): 67–80. PubMed Abstract \| Publisher Full Text\n\nShrestha R, Matteis L, Skofic M, et al.: Bridging the phenotypic and genetic data useful for integrated breeding through a data annotation using the Crop Ontology developed by the crop communities of practice. Front Physiol. 2012; 3: 326. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThe Plant Ontology Consortium: The Plant Ontology™ Consortium and Plant Ontologies. Comp Funct Genomics. 2002; 3(2): 137–142. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nThompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994; 22(22): 4673–4680. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKrajewski P, Chen D, C´wiek H, et al.: Towards recommendations for metadata and data handling in plant phenotyping. J Exp Bot. 2015; 66(18): 5417–5427. PubMed Abstract \| Publisher Full Text\n\nĆwiek-Kupczyńska H, Altmann T, Arend D, et al.: Measures for interoperability of phenotypic data: minimum information requirements and formatting. Plant Methods. 2016; 12(1): 44. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nDavey RP, Thanki AS, Bian X: Tgac browser: visualisation solutions for big data in the genomic era. 2016. Reference Source\n\nTello-Ruiz MK, Stein J, Wei S, et al.: Gramene 2016: comparative plant genomics and pathway resources. Nucleic Acids Res. 2016; 44(D1): D1133–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nGubała T, Szymczyszyn T, Nowakowski P, et al.: TGAC/brassica: v1.0.0. Zenodo. 2017. Data Source" }	[ { "id": "21825", "date": "03 May 2017", "name": "Christopher J. Rawlings", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe background to the development of the Brassica Information Portal and its origins in the CropStore system are well described. This paper is an update on the work in progress to deliver an integrated resource linking phenotype to genotype data for Brassica species.\n\nThere are welcome new developments in the BIP including greater use of ontologies, in particular links with the MIAPPE initiative to capture phenotype in a systematic way and to adopt emerging metadata standards for crop phenotyping projects.\n\nA summary of the contents demonstrates that the BIP already comprises a significant scale repository for Brassica pre-breeding data and where relevant this is linked to SNP data visible with a genome browser.\n\nThis paper is about the BIP as an information resource, rather than a publication of a new method. The paper provides a high level view of the technical implementation, reporting on adoption of the PostgreSQL relational database technology and the general use of Restful services as the basis for the data management operations in the BIP, including those that support improved interfaces for data loading and for browsing. A conceptual database schema is presented for the BIP database.\n\nThere are other areas of BIP that are adopting good practise in science data management, including the use of Orcid identifiers for user authentication and data search and retrieval using Elasticsearch technology.\n\nThe paper provides overviews of key data management tasks and gives more information in the Supplementary material provided with the manuscript. There are also links to the Open Source repository Github where the software for BIP is available and to the example code for querying the data programmatically.\n\nOverall, this is an informative paper about the current state of the Brassica Information Portal. It is not a primary research paper, but rather a report of work in progress supporting the Brassica research community with a useful resource and software that could be re-used for other crops where there is a requirement for the management and dissemination of crop genotype-phenotype datasets.\n\nWhile there has been good progress so far towards developing the software so that the system supports open access to the software and data, it remains unclear to what extent the FAIR principles of data stewardship are being embedded in the BIP software. (e.g. Wilkinson et al1). In particular, it would be helpful for the authors to describe their plans for exposing discoverability metadata and how they view the role of BIP as a “FAIRport” for Brassica data.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3193", "date": "20 Nov 2017", "name": "Wiktor Jurkowski", "role": "Author Response", "response": "Dear Prof. Rawlings,Thank you very much for your time and feedback!A new version of this manuscript has been published with the changes hopefully addressing your concerns. In particular, we added a paragraph on application of FAIR principles in BIP, current status and where BIP requires further improvements.Kind regardsWiktor Jurkowski" } ] }, { "id": "21824", "date": "05 May 2017", "name": "Cyril Pommier", "expertise": [ "Reviewer Expertise Distributed information systems", "computer science", "phenotyping data managment" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe rational and the objective of the Brassica Information Portal is well explained. Its complementarity with existing data repositories including genomic data repositories and tools and in particular its role as an important repository for Brassica Phenotyping Data is clear. Its relation to other Phenotyping Brassica repositories could be further discussed as perspectives.\nBIP makes good use of the current standards for plant phenotyping data, in particular MIAPPE and Cropontology. By being implicated in the current evolutions of MIAPPE, the BIP team demonstrate its will to ensure interoperability of this system with international standards. This is important for long term sustainability.\nThe data submission process is well described. It shows that significant efforts have been made to ensure data quality and to ease data managers task. Data consistency between population, ontologies and trait datasets should be ensured with the chosen mechanisms. Data citation is well ensured by a DOI generation for datasets.\nBIP use modern and proven technological solutions, like Elasticsearch, Nginx web server, rich BIP Web Service API backing the whole user interface and allowing user or programatic interaction with the system.\nFor the technical description of the software tool to be fully sound, the following points should be improved.\nThe role of BIP in the BrAPI should be further described. In the Author Contributions, BrAPI is cited but it doesn't appears elsewhere in the text. Some technical description could be improved, for instance \"universal search\" isn't very clear.\nThis paper shows very well the development process of the BIP information system. The current status of the system is clear. Its perspectives could be further discussed regarding FAIR compatibility, Open Data including data licences policies (CC-by). Alongside this point, BIP team curent opinion on distributed phenotyping information system, backed by the BrAPI, could be discussed.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Partly\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3194", "date": "20 Nov 2017", "name": "Wiktor Jurkowski", "role": "Author Response", "response": "Dear Dr Pommier,Thank you very much for your time and feedback!A new version of this manuscript has been published. I hope we were able to improve technical description of the resource. Kind regards,Wiktor Jurkowski" } ] }, { "id": "22091", "date": "09 May 2017", "name": "Matthew M. Nelson", "expertise": [ "Reviewer Expertise Plant breeding", "crop genetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis manuscript describes a valuable resource for the Brassica research and breeding communities. It is logically and clearly written.\n\nI felt that some of the methodology was inadequately cited. For example, on P4: the terms \"PostgreSQL RDBMS technology”, “Ruby on Rails”, “Nginx” and “Elasticsearch” are used without any external references. Ideally scholarly or technical journal articles would be cited; if not available, then the publisher's name or project website.\n\nSome minor edits required: P3: The phrase ‘assist and help’ contains a tautology. Choose one of these terms, not both. P3: Replace ‘steming’ with ‘stemming’ P6: Presumed typo in the header “0.1 Content navigation”?\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [ { "c_id": "3195", "date": "20 Nov 2017", "name": "Wiktor Jurkowski", "role": "Author Response", "response": "Dear Dr Nelson,Thank you very much for your time and feedback!A new version of this manuscript has been published with substantial number of edits (including your suggestions) throughout the manuscript that we hope improved overall quality of the paper.Kind regards,Wiktor Jurkowski" } ] } ]	1	https://f1000research.com/articles/6-465
https://f1000research.com/articles/6-223/v1	06 Mar 17	{ "type": "Research Article", "title": "Preference and willingness to pay for nutritional counseling services in urban Hanoi", "authors": [ "Hai Viet Nguyen", "Ngoc Bao Trinh", "Huong Thi Le", "Cuong Tat Nguyen", "Hue Thi Mai", "Tho Dinh Tran", "Huong Thi Le", "Bach Xuan Tran", "Thuc Thi Minh Vu", "Hai Viet Nguyen", "Ngoc Bao Trinh", "Huong Thi Le", "Hue Thi Mai", "Tho Dinh Tran", "Huong Thi Le", "Bach Xuan Tran", "Thuc Thi Minh Vu" ], "abstract": "Background: Despite substantial achievement in reducing malnutrition rates in Vietnam, there has been an increasing rate of overweight individuals in urban areas, which may result in a high burden of non-communicable diseases. Nutritional counseling clinics have been introduced in several settings; however, little is known about the preference for this service among urban clients. This study aimed to assess the preference and willingness to pay (WTP) for nutritional counseling services among urban clients. Methods: We interviewed 429 clients who attended Hanoi Medical University Nutritional Counseling Clinic (Hanoi, Vietnam). WTP was determined using double-bounded dichotomous-choice questions and open-ended questions. Results: In total, 78.6% respondents were willing to use nutritional counseling services. The mean amount of WTP for one-time service and one-year package was 96,100VND (~$4.3) and 946,400VND (~$41.9), respectively. Clients’ willingness to use the service was higher among females, those seeking counseling for elderly people and those who preferred face-to-face counseling services (p<0.05). WTP was higher among those who were over 35 years old, those seeking services for the elderly people, those having poor nutritional status, and those having under-6 year old children (p<0.05). Conclusions: The preference and WTP for nutritional counseling services in urban Hanoi were relatively high. Scaling up this service is necessary to actively prevent and control the spread of non-communicable diseases.", "keywords": [ "Demand", "willingness to pay", "WTP", "nutrition counseling services" ], "content": "Introduction\n\nIn recent years, Vietnam has achieved a significant improvement in people’s health and nutritional status1. This is indicated by an improvement in people’s knowledge, attitude and practice on nutrition, and a significant decrease in malnutrition rates among children. According to the National Institute of Nutrition, the rate of marasmus and stunting has been reduced from 19.9% (2013) to 14.1% (2015) and 32.6% (2013) to 24.6% (2015), respectively1. However, in urban areas, significant increases in overweight and obesity rates may result in high burden of non-communicable diseases (NCDs)2. A survey among 17,213 people in Vietnam showed that the rate of overweight and obesity was 16.3%. This high rate was fueled by unhealthy diet habits, alcohol abuse and sedentary lifestyles3.\n\nIn developed countries, nutritional counselling has been recognized as an effective measure to improve awareness and encourage a healthy lifestyle, and has been shown to reduce the risk of obesity and NCDs4. Nutritional counseling clinics can be organized in co-location with other general health care services or as stand-alone sites. However, in resource-scarce settings, this model has not yet to be implemented widely, due to the low responsiveness of health systems, as well as the poor practice of prevention against nutrition-related problems among the population5. This condition can be seen in several countries around the world, such as Denmark or Western Australia5–7.\n\nIn Vietnam, nutritional counseling clinics have been recently introduced in metropolitan areas, including Hanoi and Ho Chi Minh City. However, little is known about the profile and preference of the clients that attend these clinics. To inform policy development and support the expansion of this service, the present study was conducted to assess the preference and willingness of clients to pay for nutritional counseling services in an urban site in Hanoi.\n\n\nMethods\n\nA cross-sectional study was conducted from March to April 2016 in an urban clinic in Hanoi Medical University, Hanoi, Vietnam. Eligibility criteria included 1) clients attending services in the Center of Preventive Medicine at Hanoi Medical University; and clients’ parents or guardians (for those who were under 18 years old); 2) aged 18 years and above; 3) agreed to participate in this study and gave written informed consent; 4) able to answer a questionnaire (Supplementary File 1 and Supplementary File 2) for 15–20 minutes.\n\nAll eligible respondents from March to April 2016 were invited to participate in the study, resulting in a sample size of 429.\n\nSocio-demographic variables included age, gender, ethnicity, religion, educational attainment, marital status, current occupation, self-assessment of nutritional status and monthly household income (see Table 5 for detail).\n\nPreference for nutritional counseling services included who would receive nutritional counseling, frequency of counseling services and communication methods for counseling.\n\nWillingness to pay for nutritional counseling services were elicited using the bidding game technique, which consists of double-bounded dichotomous-choice questions combined with an open-ended question regarding two service packages: 1) fee-for-service; and 2) one-year nutritional management package.\n\nWe selected 200,000 VND (~ US$ 9; 2017 exchange rate) and 3,000,000 VND (~ US$ 135; 2017 exchange rate) to be the initial prices for fee-for-service and one-year nutritional management package, respectively, based on the actual price of nutritional counseling services in this clinic. Each patient was asked a series of questions about their WTP at specific prices (see Figure 1 and Figure 2 for the bidding process). Firstly, the clients were asked if they were willing to pay the initial prices. Depending on the choice of either Yes or No, interviewers presented two other bids: the higher bid for respondents answering “Yes”; and the lower bid for respondents saying “No”. The question was repeated until the last bid was equal to four times or one eighth of the initial prices. Finally, the respondents were asked an open-ended question “What is the maximum price you would be willing to pay for nutritional counseling services?”\n\nData was analyzed using STATA software version 12.0 (Stata Corp. LP, College Station, TX, USA). A p-value <0.05 was considered statistically significance. A stepwise logistic model with the threshold of p-value < 0.2 was used to identify associated factors with the WTP. Interval regression was used to measure the amount of WTP and identify associated factors.\n\nProposal of this study was approved by the Ethical Committee of Hanoi Medical University. Subjects were introduced to the purpose of this study, and asked to give written informed consent if they agreed to participate in the study. Respondents could withdraw anytime they want. Their information was ensured to be confidential.\n\n\nResults\n\nDemographic and socio-economic statuses of respondents are summarized in Table 1. Most of the clients were Kinh (97.7%), having above high school education (63.2%), single with no children (50.6–60.0%), and were in white collar employment (43.3%).\n\n* Adults with children that were <18 years old.\n\n** Some respondents refused to provide characteristic information, resulted in missing values.\n\nTable 2 shows the willingness to use for nutritional counseling services of clients. Overall, 79.6% clients wanted to use counseling services. The major desire was that respondents’ children would receive nutritional counseling (74.8%) monthly or more frequently (39.8%) via meeting physicians face-to-face (64.9%).\n\n* Adults with children that were <18 years old.\n\nThe WTP for one-time service is described in Table 3. Overall, a high amount of the respondents were willing to pay for nutritional counseling services (87.2%). The mean amount they were willing to pay was 96,100 VND per utilization (95% CI 81,000–111,000 VND), equivalent to US $4.3 in 2017, which varied across groups. There was a significant difference in the WTP of the three age groups (p<0.05).\n\naPercentage of 297 clients who responded to one-time service questions.\n\nbTwo clients’ employment statuses were missing.\n\nTable 4 describes the WTP for the one-year nutrition management package. On average, respondents were willing to pay 946,400VND (95% CI 860,200 – 1,032,700 VND) (~$41.9 - 2017) for this package, which varied among groups (p<0.05).\n\naPercentage of 372 clients who responded to one-year package questions.\n\nbOne client’s employment status was missing.\n\nAssociated factors of the willingness to use and WTP for nutritional counseling services are shown in Table 5. The likelihood of using nutritional counseling services was higher among females, those seeking counseling for elderly people and those that preferred face-to-face counseling services. WTP for one-time service was 95,000 VND higher among clients aged over 35. Meanwhile, WTP for one-year nutritional management services was higher among those seeking services for the elderly people, those with a poor nutritional status and those that have under-6 year old children.\n\n* p<0.01, p<0.05, * p<0.1\n\naHousehold income: Poorest, ≤7,000,000VND/month (~$307.4); Poor, 7,000,000 – 10,000,000VND/month (~$307.4 – $439.2); Average, 10,000,000 – 15,000,000VND/month (~$439.2 – $658.8); Rich, 15,000,000 – 20,000,000VND/month (~$658.8 – $878.3); Richest, >20,000,000VND/month (~$878.3).\n\nbNutritional status (self-assessment of respondents), including: Very good; Good; Average; Poor; Very poor.\n\n\nDiscussion\n\nNutrition has been a pressing topic of many researchers8. There are several studies about nutritional counseling services for patients9–11 or concerning a particular nutritional component12,13, but studies about general and preventive nutritional counseling are still limited14. Evidence provided by this study not only imparts information for future research, but also gives nutritional counseling providers a better perception to enhance their services.\n\nIn this urban setting, we found a high preference for nutritional counseling services for various target client groups, including elderly people and children. Clients also reported a high WTP for this service, which could be very helpful for expansion of the services. However, a combination of communication methods is needed; we found a higher preference for face-to-face counseling among respondents, knowing that many of them may also seek other health care services.\n\nOverall, the preference for nutritional counseling in this study was quite high (79.6%). Most of the clients who did not have the need for this service were single with no children and self-evaluated their nutritional status as ‘average’. The mean amount of WTP for one-time and one-years services was $4.3 and $41.9, accounting for 0.20% and 1.98% GDP per capita in Vietnam in 2015 ($2,111, enumerated by World Bank)15, which is an acceptable amount for clients to pay.\n\nAssociated factors of the preference and WTP for nutritional counseling services in our study were not in line with some predictions provided by a study in South Korea16. Our study showed that older clients are more willing to pay for nutritional counseling than younger ones. Another noteworthy finding of this study is that clients with a higher educational level were not as willing to pay for the one-year management package as clients who only finished high school. This can be explained by the two occupations of respondents: those whose educational level were above high school were mainly white-collar workers, while almost everyone with lower educational levels were still high-school students or college students (83.8%). This may suggest that the recent nutritional education programs in Vietnam have caused a positive effect on students’ attitude toward nutritionally related programs (http://dinhduonghocduong.net/)17.\n\nThose who have under-6 year old children and assess their children’s nutrition status poorly had a higher WTP for nutritional counseling services. These findings are well expected, thus enhance our study data’s validity. We suspected that clients’ income was associated with their WTP, as richer clients are more likely to pay a higher amount for nutritional counseling services. However, there was no significant relationship between clients’ household income and the WTP for nutrition counseling services.\n\nTo elicit a clients’ preference and WTP, we used the bidding game technique, as it was proved to be more reliable than open-ended questions or dichotomous-choice questions only18,19. However, one of the biggest drawbacks of this technique is that the risk of starting-point bias - the initial bid can have influence on clients’ WTP20. The initial bids in this study were based on the actual prices for nutritional counseling services in this setting in order to minimize the occurrence of this bias. Additionally, our study may possibly be affected by other biases, such as observation bias, which occurs when the roles of respondents in their families can affect the amount of their WTP21. For example, we assumed that those who were the bread-winners in their families tended to have higher WTP for health-related services. Another example is that if information about nutritional counseling may not be sufficiently provided, this may result in lower preference and WTP for nutritional counseling services. To mitigate this bias, we selected highly-experience interviewers and trained them carefully with a standardized protocol for data collection.\n\n\nConclusions\n\nThe preference and willingness to pay for nutritional counseling services in urban Hanoi is relatively high. These findings may partly contribute to the implementation of maintaining nutritional counseling services Vietnam, thus actively preventing and controlling the spread of non-communicable diseases.\n\n\nData availability\n\nDataset 1: Raw data for Table 1–Table 5. doi, 10.5256/f1000research.10974.d15326022", "appendix": "Author contributions\n\n\n\nNBT, HTL and BXT designed the study. All authors undertook data collection and analysis. HVN wrote the first draft. All authors approved the final version to be published.\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgements\n\nWe would like to thank Hanoi Medical University (HMU) and HMU Vaccination Clinic for their support for this study.\n\n\nSupplementary material\n\nSupplementary File 1: Questionnaire in English.\n\nClick here to access the data.\n\nSupplementary File 2: Questionnaire in Vietnamese.\n\nClick here to access the data.\n\n\nReferences\n\nPrevalence of Malnutrition in Vietnamese children under 5 years old. Vietnam National Institute of Nutrition. 2015.\n\nMathers CD, Loncar D: Projections of global mortality and burden of disease from 2002 to 2030. PLoS Med. 2006; 3(11): e442. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nOverweight - Obesity and associated factors of 25–64 years old Vietnamese. Vietnam National Institute of Nutrition. 2011.\n\nWillaing I, Ladelund S, Jørgensen T, et al.: Nutritional counselling in primary health care: a randomized comparison of an intervention by general practitioner or dietician. Eur J Cardiovasc Prev Rehabil. 2004; 11(6): 513–520. PubMed Abstract \| Publisher Full Text\n\nWillaing I, Ladelund S, Jørgensen T, et al.: Nutritional counselling in primary health care: a randomized comparison of an intervention by general practitioner or dietician. Eur J Cardiovasc Prev Rehabil. 2004; 11(6): 513–20. PubMed Abstract \| Publisher Full Text\n\nKushner RF: Barriers to providing nutrition counseling by physicians: a survey of primary care practitioners. Prev Med. 1995; 24(6): 546–52. PubMed Abstract \| Publisher Full Text\n\nPritchard DA, Hyndman J, Taba F: Nutritional counselling in general practice: a cost effective analysis. J Epidemiol Community Health. 1999; 53(5): 311–316. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nFischer AR, Berezowska A, van der Lans IA, et al.: Willingness to pay for personalised nutrition across Europe. Eur J Public Health. 2016; 26(4): 640–4. PubMed Abstract \| Publisher Full Text\n\nHuang MA, Greenson JK, Chao C, et al.: One-year intense nutritional counseling results in histological improvement in patients with non-alcoholic steatohepatitis: a pilot study. Am J Gastroenterol. 2005; 100(5): 1072–1081. PubMed Abstract \| Publisher Full Text\n\nOlsen J, Willaing I, Ladelund S, et al.: Cost-effectiveness of nutritional counseling for obese patients and patients at risk of ischemic heart disease. Int J Technol Assess Health Care. 2005; 21(2): 194–202. PubMed Abstract\n\nEnglish RM, Badcock JC, Giay T, et al.: Effect of nutrition improvement project on morbidity from infectious diseases in preschool children in Vietnam: comparison with control commune. BMJ. 1997; 315(7116): 1122–5. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKim J, Lee Y, Kye S, et al.: Association of serum vitamin D with osteosarcopenic obesity: Korea National Health and Nutrition Examination Survey 2008-2010. J Cachexia Sarcopenia Muscle. 2016. PubMed Abstract \| Publisher Full Text\n\nVidailhet M, Rieu D, Feillet F, et al.: Vitamin A in pediatrics: An update from the Nutrition Committee of the French Society of Pediatrics. Arch Pediatr. 2017; pii: S0929-693X(16)30604-2. PubMed Abstract \| Publisher Full Text\n\nNguyen PH, Hoang MV, Hajeebhoy N, et al.: Maternal willingness to pay for infant and young child nutrition counseling services in Vietnam. Glob Health Action. 2015; 8(1): 28001. PubMed Abstract \| Publisher Full Text\n\nBank TW: GDP per capita (current US$). Vietnam, 2015.\n\nEngelhardt K, Ahn BC, Cho SI, et al.: Predictors of interest in nutrition topics and willingness to participate in local nutrition programmes. J Public Health (Oxf). 2007; 29(1): 9–12. PubMed Abstract \| Publisher Full Text\n\nNutrition Education at school. Vietnam National Institute of Nutrition. 2011.\n\nDong H, Kouyate B, Cairns J, et al.: A comparison of the reliability of the take-it-or-leave-it and the bidding game approaches to estimating willingness-to-pay in a rural population in West Africa. Soc Sci Med. 2003; 56(10): 2181–9. PubMed Abstract \| Publisher Full Text\n\nO'brien B, Viramontes JL: Willingness to pay: a valid and reliable measure of health state preference? Med Decis Making. 1994; 14(3): 289–97. PubMed Abstract \| Publisher Full Text\n\nMilanesi J: Measuring demand for sanitation in developing countries: A new theoretical and methodological framework for contingent valuation surveys.12th Conference of the Association for Heterodox Economics. 2010. Reference Source\n\nKanchanaraksa S: Bias and Confounding. Johns Hopkins University. Reference Source\n\nNguyen HV, Trinh NB, Le HT, et al.: Dataset 1 in: Preference and willingness to pay for nutritional counseling services in urban Hanoi. F1000Research. 2017. Data Source" }	[ { "id": "20740", "date": "09 Mar 2017", "name": "Quan-Hoang Vuong", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis research study has been well documented and technically sound, with regard to statistical consideration. The analysis employs standard techniques and sample size has been reasonable (N=429). The report of results should be welcome as the need for healthcare and related data has become increasingly imperative.\nThe authors of the work have presented interesting findings, especially the significance of a small amount of spending, roughly $4 for one-time counseling service, and $42 for one-year package of services, both significant at conventional statistical levels. Apparently, there are rooms for the authors to report more insights since I can see the opportunity for controlling for some of the most influential socio-economic demographic factors, such as income, or physical state, such as BMI, where conditional probabilities computed from appropriate modeling efforts can potentially be insightful.\nI was also a bit surprised with the fact that the rate of approval for use of ICT apps and devices has been quite low, and this reality will be in and of itself an issue worth exploring as they are critically important in today's age of information, and help reduce counseling costs.\nAll in all, this is a good and useful study, and I am happy to approve it for indexing by F1000Research.", "responses": [] }, { "id": "20978", "date": "29 Sep 2017", "name": "Wongsa Laohasiriwong", "expertise": [ "Reviewer Expertise Health policy", "heath service system", "epidemiology", "spatial epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe first, the topic is relevant and interesting. However, the authors need to clarify some points for future replication by other.\n\nPlease confirm code of the dependent variable and independent variables in this study.I am not sure whether the dependent variable is Preference or WTP1 or WTP2.I try to replicate the result by using STATA program, but the result of the multiple logistic regressions is not the same those presenting in Table 5.\n\nMy analysis are:\n\nTable 1 Table 2 Table 3\n\nRemarks on Table 5 mentions about the Vietnamese economic status using the monthly income quintile. It would help us gaining better understanding if the authors discuss how the amount of 2,000,000 VND for single service and 3,000,000 VHD for yearly services related to their income status. Furthermore, if it is calculated as % of total expense, we will have better understanding on whether it is considered as high cost, or catastrophic cost or not. However, for sure the expense for this counseling service is not too high. How could we guarantee that this could reflect the real perception of the value of counselling as priority and their willingness to pay when compare to other services such as treatment that have better short term output or outcomes.\nThe methodology of biding to get the WTP amount is very interesting that the participants choose the amount themselves and use their mean as WTP. Please discuss concerning the amount that finally identify as WTP is appropriate in the real context of their living standard.\n\nParticipants of this research are clients who are seeking services at the Center of Preventive Medicine. Researcher should explain whether the sample could represent to population, since they might have some conditions that have higher demand for the services, then the results might be over estimate.\nSince this study is interesting:\nIs the nutrition counseling fee included in their service package (with all service) that they must pay. Or The nutrition counseling is separate service that they could select or not select?\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Partly\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [ { "c_id": "3076", "date": "02 Oct 2017", "name": "Cuong Tat Nguyen", "role": "Author Response", "response": "Dear Prof. Wongsa Laohasiriwong,Thank you very much for your review. I will provide explanations for your questions.1. Both Preference, WTP1, and WTP2 are dependent variables, as they represented clients' willingness to use the nutritional counseling service, willingness to pay for one-time service and for a one-year package.2. We selected 200,000 VND and 3,000,000 VND to be the initial prices for the one-time service and one-year package, respectively, as they were the actual prices of nutritional counseling services in this clinic at that time. These prices were accounted for 0.43% and 6.4% of GDP per capita in Vietnam in 2015, yet still acceptable prices for Vietnamese clients to pay.As for the comparison between this service and treatment, it is quite a hard question, since comparing the outcomes of preventive medicine and curative medicine is not an easy task. This could be a good idea for further studies about nutritional counseling service's outcome in the future.3. The mean amounts of WTP for both one-time service and one year package are suitable for the living standard of Hanoi people, as this research was carried out in an urban area of Hanoi, with the majority of participants were students and white collar workers. These prices were also relevant to other medical services' prices such as out-patient examination (~150,000 VND) or chest X-ray (~80,000 VND).4. We also have taken into account the risk of overestimating the preference of clients for nutritional counseling service, but this was not significant, as the participants were clients who sought other medical services in this clinic, e.g vaccination. The nutritional counseling service was a separate service and was not included in their medical packageSincerely,Cuong" } ] }, { "id": "24849", "date": "30 Oct 2017", "name": "Danielle Gallegos", "expertise": [ "Reviewer Expertise nutrition and dietetics" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThank you for the opportunity to review this paper. Studies of this kind are vitally important as Viet Nam builds its infrastructure for the treatment and management of NCDs. The paper is interesting and technically sound. There is scope to expand the statistical analyses to control for a range of factors but only if these data were collected for example, BMI, diagnosis of an NCD, whether the child was underweight or overweight.\n\nThe only suggestion I have is to add to the discussion regarding the recent moves to train an expanded workforce in nutrition and dietietics. HMU is the first university to run the Bachelor of Nutrition degree and this expanded workforce will in the future build capacity for the delivery of nutrition counselling services in a range of settings. Knowing that clients are willing to pay for the service will contribute significantly to sustainability. In addition, some discussion or speculation on whether this would negate the contribution of universal medical insurance would contribute to the discussion.\n\nWhile I know that the review is meant to focus on the scientific content there are some content issues that need to be addressed Abstract: I would be more general with the Dong described 96100 is too specific for the analysis performed and would be better cited as 96000 add in USD to the $ amount quoted.\npage 3 Western Australia is not a country and so this sentence needs to be adjusted.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nYes\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-223
https://f1000research.com/articles/6-33/v2	17 Jan 17	{ "type": "Research Note", "title": "A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling", "authors": [ "Dmitrii M. Nikolaev", "Anton Emelyanov", "Vitaly M. Boitsov", "Maxim S Panov", "Mikhail N. Ryazantsev", "Dmitrii M. Nikolaev", "Anton Emelyanov", "Vitaly M. Boitsov", "Maxim S Panov" ], "abstract": "It was demonstrated in recent studies that some rhodopsins can be used in optogenetics as fluorescent indicators of membrane voltage. One of the promising candidates for these applications is archaerhodopsin-3. However, the fluorescent signal for wild-type achaerhodopsin-3 is not strong enough for real applications. Rational design of mutants with an improved signal is an important task, which requires both experimental and theoretical studies. Herein, we used a homology-based computational approach to predict the three-dimensional structure of archaerhodopsin-3, and a Quantum Mechanics/Molecular Mechanics (QM/MM) hybrid approach with high-level multireference ab initio methodology (SORCI+Q/AMBER) to model optical properties of this protein. We demonstrated that this methodology allows for reliable prediction of structure and spectral properties of archaerhodopsin-3. The results of this study can be utilized for computational molecular design of efficient fluorescent indicators of membrane voltage for modern optogenetics on the basis of archaerhodopsin-3.", "keywords": [ "optogenetics", "archaerhodopsin", "protein structure prediction", "QM/MM", "spectral tuning in rhodopsins" ], "content": "Introduction\n\nPrecise and quick control of physiological processes using integrated optical and genetic methods is a vast area with a high number of important applications1. One possible approach in this field is using fluorescent voltage-dependent indicators for detecting the activity of mammalian neurons, which allows achievement of the precision of a single neuron without perceptible time delays. It was recently shown that some rhodopsins, especially achaerhodopsin-3, can be potential candidates for such a task2–5. However, the fluorescent signal for these rhodopsins is not strong enough for real applications, but insertion of specific mutations into these proteins can dramatically improve the signal quality2–5.\n\nUnfortunately, the fundamental mechanisms underlying the processes that determine fluorescence are not well understood. This lack of knowledge leads to difficulties with design of desired rhodopsin mutants. While rational design is not based on a solid foundation and, for this reason, is not very effective, another experimental approach, random mutagenesis, is very time consuming. Computational studies can provide additional insights into the problem. One of the main obstacles for computational modeling of proteins is the absence of three-dimensional structures of high quality, especially for membrane proteins, which are a challenge for crystallization. On the other hand, computational prediction of three-dimensional structures is not trivial. The goal of this study was to obtain a good-quality structure for achaerhodopsin-3, one of the most used voltage-dependent fluorescent sensors4,5, and based on this structure to predict the optical properties of this protein.\n\nTo achieve this goal, we used a homology-based computational approach for structure prediction. As the choice of a structure prediction algorithm is not straightforward, we tested several methods. To evaluate the quality of obtained structures we performed subsequent Quantum Mechanics/Molecular Mechanics (QM/MM) calculations of absorption maxima and compared the results with available experimental data.\n\n\nMethods\n\nThe structure of archaerhodopsin-3 was built using a homology modeling approach. Primary structures for all rhodopsins with crystallographic data are available in the Protein Data Bank library6 (24 structures as of September 2016) were compared with the primary structure of archaerhodopsin-3. Archeorhodopsin-1, which has the highest sequence identity to the target protein, was chosen as a template (RCSB code, 1UAZ). Three algorithms of homology-based model building were tested: Medeller7, I-TASSER8 and RosettaCM9. All methods of homology modeling heavily rely on externally made target-template alignment of primary sequences, which serves as the main instruction for model building. For this reason, we tested three algorithms of pairwise alignment using their results as an input for each method of model building. Two of the alignment methods are specifically constructed for membrane proteins, MP-T10 and AlignMe11, and the third one, MUSTER12, gains its quality from evolutionary predictions. The latter algorithm is a built-in algorithm of I-TASSER suite and its results were used only for this method of structure prediction.\n\nBefore QM/MM calculations, several preparation steps were performed: hydrogen atoms were added using pdb2pqr package13 version 2.1.1 using CHARMM force field version 2714, pH=7; hydrogen atoms were equilibrated by energy minimization in NAMD package15, version 2.11. The retinal chromophore was bound to the lysine residue Lys226, whole lysine + retinal system was parameterized in CHARMM force field16. The protein was inserted in the POPC membrane17, the whole system was inserted in a water solvent box, the TIP3P water model18 was used, the size of water box was selected so that there were at least 10 Å from any atom of protein to the edge of the system, and the system was neutralized by addition of Na+ and Cl- ions.\n\nRelaxation of the system was performed in several steps: relaxation of retinal + lysine complex with all other atoms fixed, relaxation of all atoms that were within 6 Å of the chromophore system, relaxation of whole protein and water box. During all these steps the following parameters were used: a 10 Å cutoff with switching starting at 8.5 Å was applied to the electrostatics and van der Waals interactions; Particle Mesh Ewald method19 was used for dealing with electrostatics interactions, grid spacing 1Å. Equilibrated protein structure was extracted; internal waters were added into protein cavities using WaterDock program20.\n\nTo calculate absorption maxima, we used the methodology that has been proven as efficient in a number of our previous studies for different kind of rhodopsins and rhodopsin mimics21–27. The structures of the archaerhodopsin-3 obtained at the previous step were optimized using two-layer ONIOM (QM:MM-EE) scheme. (QM=B3LYP/6-31G; MM= AMBER for aminoacids and TIP3P for water, EE=electronic embedding) was implemented in the Gaussian09 package28. To calculate the spectral properties of the chromophore in the presence of the protein environment (described as AMBER point charges) SORCI+Q/6-31G level of the theory was used, as it is implemented in ORCA6.0 package29. For details of previously performed QM/MM methodology see Altun et al.30,31.\n\n\nResults\n\nWe obtained seven structures of archaerhodopsin-3 and chose the best one by comparison of experimental spectral data with the calculated one. The results of the calculation of absorption maximum wavelength are all in the range of 31 nm from the experimental value (556 nm). They are presented in Table 1. From these results we can conclude that the best matching result was obtained using I-TASSER suite with AlignMe24 alignment (Figure 1).\n\n\nConclusions\n\nIn this study, we predicted the structure of fluorescent voltage-dependent sensor achaerhodopsin-3 and evaluated its quality with subsequent QM/MM high level ab initio calculations of spectral properties. The calculated absorption maximum is within 31 nm from the experimental value. Several methods of model building were tested and spectral characteristics were calculated for all resulting models. We showed that our methodology allowed for reliable prediction of optical properties of archaerhodopsin-3. The results of this study can be utilized for high-level QM/MM investigation of different aspects of photochemistry of this voltage-dependent fluorescent sensor and, therefore, to contribute in development of the efficient molecular tools for modern optogenetics.\n\n\nData availability\n\nThe sequence of archaerhodopsin-3 was taken from Uniprot database: http://www.uniprot.org/uniprot/P96787\n\nThe template for homology modeling was taken from PDB database (rcsb code 1UAZ): http://www.rcsb.org/pdb/explore/explore.do?structureId=1UAZ\n\nThe input files of I-TASSER suite, RosettaCM, Medeller algorithms with corresponding README files, zipped output of I-TASSER suite, scripts for processing structure after homology modeling stage (with instructions in README file), input files for spectra calculations are available: doi, 10.5281/zenodo.22916832 (https://zenodo.org/record/229168#.WG5jyFWLTcs)", "appendix": "Author contributions\n\n\n\nMNR proposed and designed the research, DMN, MSP and MNR made the calculations. AE and VMB provided valuable consultation during research. MNR and DMN wrote the paper\n\n\nCompeting interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nMNR were supported by Russian Foundation for Basic Research (grant numbers, 14-04-01339 A and 15-29-03872 ofi_m).\n\n\nReferences\n\nDeisseroth K: Optogenetics. Nat Methods. 2011; 8(1): 26–29. PubMed Abstract \| Publisher Full Text\n\nEngqvist MK, Mclsaac RS, Dollinger P, et al.: Directed evolution of Gloeobacter violaceus rhodopsin spectral properties. J Mol Biol. 2015; 427(1): 205–220. PubMed Abstract \| Publisher Full Text\n\nKralj JM, Hochbaum DR, Douglass AD, et al.: Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science. 2011; 333(6040): 345–348. PubMed Abstract \| Publisher Full Text\n\nKralj JM, Douglass AD, Hochbaum DR, et al.: Optical recording of action potentials in mammalian neurons using a microbial rhodopsin. Nat Methods. 2012; 9(1): 90–95. 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Publisher Full Text\n\nSchapiro I, Ryazantsev MN, Frutos LM, et al.: The ultrafast photoisomerizations of rhodopsin and bathorhodopsin are modulated by bond length alternation and HOOP driven electronic effects. J Am Chem Soc. 2011; 133(10): 3354–3364. PubMed Abstract \| Publisher Full Text\n\nSumita M, Ryazantsev MN, Saito K: Acceleration of the Z to E photoisomerization of penta-2,4-dieniminium by hydrogen out-of-plane motion: theoretical study on a model system of retinal protonated Schiff base. Phys Chem Chem Phys. 2009; 11(30): 6406–6414. PubMed Abstract \| Publisher Full Text\n\nFrisch MJ, Trucks GW, Shelegel HB, et al.: gaussian 09, Gaussian, Inc., Wallingford, CT 4, 2009.\n\nNeese FJ: A spectroscopy oriented configuration interaction procedure. J Chem Phys. 2003; 119(18): 9428–9443. Publisher Full Text\n\nAltun A, Yokoyama S, Morokuma K: Spectral tuning in visual pigments: an ONIOM(QM:MM) study on bovine rhodopsin and its mutants. J Phys Chem B. 2008; 112(22): 6814–6827. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAltun A, Yokoyama S, Morokuma K: Mechanism of spectral tuning going from retinal in vacuo to bovine rhodopsin and its mutants: multireference ab initio quantum mechanics/molecular mechanics studies. J Phys Chem B. 2008; 112(51): 16883–16890. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nNikolaev DM, Emelyanov A, Boitsov VM, et al.: Supplementary information for: \"A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling\" [Data set]. Zenodo. 2017. Data Source" }	[ { "id": "19223", "date": "31 Jan 2017", "name": "Kirill A. Afonin", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nOverall the paper by Nikolaev et al., is well written and an interesting part of the photo- and biochemical realm. The paper provides very promising results and a unique way of analyzing structure functionality. I am interested to see what other results arise and the how the applications indicated can be strengthened. I have just a couple of minor suggestions that may help to understand the described subject a bit better.\nIn your abstract please name some examples of real applications.\n\nThe methodology and computational analysis was a little over my head and difficult to follow. However, the proper audience for this paper may be for those with a stronger foundation of computational analysis and programs indicated in the paper.\n\nArchaerhodopsin-1 is misspelled on page 2.\n\nBest wishes to this group and any further work that will be done!", "responses": [] }, { "id": "20866", "date": "20 Mar 2017", "name": "Adam E. Cohen", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nNikolaev et al. attempt to predict archaerhodopsin-3 structural and optical properties using in silico approaches, layering QM/MM calculations of absorption spectra over homology based structural modeling. As the authors note, such work would be invaluable for guiding the design of new archaerhodopsin variants, with potential broad applicability in the fields of optogenetics and voltage imaging.\nThe creation of a ground-state homology model is a useful first step, but the authors should acknowledge and discuss some of the challenges that would need to be addressed for this model to be useful in predicting voltage-sensitive fluorescent properties. These are:\nThe fluorescence of wild-type Arch-3 appears not to come from the ground state, but from a photogenerated intermediate (see1). Thus a model would need to adopt the correct retinal isomerization state and opsin conformation to reproduce the fluorescence.\n\nTo predict voltage sensitivity, a model would need to incorporate the intra-membrane electric field and its influence on the motion of protons within the structure.\n\nThe fluorescence quantum yield depends on the branching ration in the electronic excited state between radiative and non-radiative decay pathways. A prediction of quantum yield would need to include simulation of these excited state dynamics.\nThere are several other ways in which the manuscript could be improved:\nThe abstract states, “the fluorescent signal for wild-type achaerhodopsin-3 is not strong enough for real applications.” The first paragraph repeats this claim. This is an overly pessimistic view. Many papers have used wild-type Archaerhodopsin 3 (note spelling) and its mutants for fluorescent voltage imaging. A more accurate statement would be, “An increase in the fluorescence of Archaerhodopsin 3 would broaden the scope of its possible applications.”\n\nArch-1 was used as a homology model for Arch-3.How similar are the two proteins? What percentage sequence identity and sequence homology?\n\nThe sole figure shows the best output structure, but doesn’t compare it to anything. It should at least be superimposed with the Arch-1 input structure. The outputs of the different alignment methods should also be compared and any differences highlighted. It would also be useful to have more details on where the Arch-1 and Arch-3 sequences differ - perhaps a linear sequence alignment and a figure showing where differences occur in the 3-D structure.\n\nThe authors reference several papers which have performed site-directed mutagenesis on this protein. They should highlight those residues on their output structure. Ideally, they should also perform their AlignMe/I-TASSER analysis on one of the well characterized mutants to show if their model has predictive value.\n\nFor scientists unfamiliar with the possibilities of in silico modeling, it is difficult to evaluate whether an 18 nm deviation in absorbance maximum is a lot or a little. It would be helpful to give context using other, previously studied scaffolds. How accurately can the absorbance maximum of eGFP be determined? Of another membrane protein, such as channelrhodopsin-2?\n\nThe authors admirably package and make available all of the inputs and outputs of their calculations. Unfortunately, the abstruse organization of the data makes it difficult to extract useful information from their data. The root directory of the supplemental data should include a single clearly written readme.txt file explaining how to access the output structures of each of their homology models. The output files should be obviously labeled - meaning “Figure1structure” not “P96787_wi” - and be in a commonly used file format, such as .pdb.", "responses": [ { "c_id": "3176", "date": "15 Nov 2017", "name": "Mikhail N. Ryazantsev", "role": "Author Response", "response": "Dear Prof. Cohen and Samouil Farhi. Thank you for your review of our article “A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling”. We have carefully read it and made the required changes in the article.1. The abstract states, “the fluorescent signal for wild-type achaerhodopsin-3 is not strong enough for real applications.” The first paragraph repeats this claim. This is an overly pessimistic view. Many papers have used wild-type Archaerhodopsin 3 (note spelling) and its mutants for fluorescent voltage imaging. A more accurate statement would be, “An increase in the fluorescence of Archaerhodopsin 3 would broaden the scope of its possible applications.”To refer to this comment, we added the following changes into the abstract:“One of the promising candidates for these applications is archaerhodopsin-3. While it has already shown encouraging results, there is still a large room for improvement. One of possible directions is increasing the intensity of the protein's fluorescent signal.”In the first paragraph we made the similar changes:“While the results obtained for archaerhodopsin-3 are already very encouraging, increasing the fluorescent signal of such proteins can lead to a significant progress in this field.”2. “Arch-1 was used as a homology model for Arch-3.How similar is the two proteins? What percentage sequence identity and sequence homology?”Arch-1 and Arch-3 are very evolutionally close. We added this information in the “Methods” section of the new version of our article: “sequence identity 84.5%, sequence similarity 91.5%”.3. “The sole figure shows the best output structure, but doesn’t compare it to anything. It should at least be superimposed with the Arch-1 input structure. The outputs of the different alignment methods should also be compared and any differences highlighted. It would also be useful to have more details on where the Arch-1 and Arch-3 sequences differ - perhaps a linear sequence alignment and a figure showing where differences occur in the 3-D structure.”To refer to this comment, we edited the “Results” section of our article. We added the comparison of outputs of the three alignment methods we used in our work. We showed that the results of AlignMe and MUSTER algorithms are identical, and they only slightly (in the beginning of the sequences) differ from the MP-T alignment results (Figure 1, a-c in the article). We added the following part into our article:“The results of AlignMe and MUSTER algorithms were identical; they differ from the MP-T alignment result in the flexible N-terminal part (Figure 1, a-c).”We also compared the crystallographic structure of archaerhodopsin-1 and the predicted model of archaerhodopsin-3 (Figure 2, 3 in the article). We showed that the structures of two proteins are very similar, and they differ only in loop regions and on the edges of alpha-helices, i.e. in the regions located quite far from the chromophore. We added the following text in the “Results” section of our article:“Comparison of crystallographic structure of archaerhodopsin-1 with the predicted structure of archaerhodopsin-3 showed that these two proteins differ only in loop regions and on the edges of the alpha-helices (Figure 2, 3). These regions are located on relatively large distance from the retinal chromophore.”4. “The authors reference several papers which have performed site-directed mutagenesis on this protein. They should highlight those residues on their output structure. Ideally, they should also perform their AlignMe/I-TASSER analysis on one of the well characterized mutants to show if their model has predictive value.”We highlighted the residues, which were the sites for the site-directed mutagenesis of the archaerhodopsin-3 (Mclsaac RS et al, PNAS, 2014) (Figure 4 in the article). The computational characterization of mutants is an interesting problem and we will consider it in our research. We hope to publish an article about computational investigation of different mutants of both archaerhodopsin-3 and Gloebacter violaceus rhodopsin.We added the following text in the “Results” section of our article:“We also highlighted the residues, which were the sites for the site-directed mutagenesis of the archaerhodopsin-35 (Figure 4). The computational characterization of these mutants is an interesting problem for further investigations.” 5. “For scientists unfamiliar with the possibilities of in silico modeling, it is difficult to evaluate whether an 18 nm deviation in absorbance maximum is a lot or a little. It would be helpful to give context using other, previously studied scaffolds. How accurately can the absorbance maximum of eGFP be determined? Of another membrane protein, such as channelrhodopsin-2?” In this study, all models provide reasonable results for the values of absorption maxima. Considering the approximations we use in the QM/MM methodologies, the deviation range up to 40 nm is considered to be good; however, in complicated cases we can obtain a 50-60 nm shift from the experimental value. Modeling of the absorption maxima is very sensitive to the quality of three-dimensional structures, and even relatively small changes of the structures can lead to huge shifts of this value.In the case of the microbial rhodopsins we obtained many results for the absorption maxima of proteins with available crystallographic structure. For example, we obtained a 29 nm shift for halorhodopsin from N. pharaonis, a 25 nm shift for archaerhodopsin-2 and a 39 nm shift for the channelrhodopsin-2. These results lie in the same deviation range, and for this reason we think that the models we obtained have quite high quality. Substantial errors in the models would lead to much bigger shifts (up to 100-200 nm).Considering these remarks, we added the following modifications in the “Results” section:“This deviation range is sensible considering the approximations used in our methodologies. In our previous studies, we obtained a 29 nm shift for the absorption maximum of halorhodopsin from N.pharaonis23, a 25 nm shift for the absorption maximum of archaerhodopsin-2 and a 39 nm shift for the channelrhodopsin-2. The model with the smallest deviation was predicted by I-TASSER algorithm with AlignMe alignment (Figure 2).” 5. “The authors admirably package and make available all of the inputs and outputs of their calculations. Unfortunately, the abstruse organization of the data makes it difficult to extract useful information from their data. The root directory of the supplemental data should include a single clearly written readme.txt file explaining how to access the output structures of each of their homology models. The output files should be obviously labeled - meaning “Figure1structure” not “P96787_wi” - and be in a commonly used file format, such as .pdb.”Considering this remark, we created a new version of the archive:https://zenodo.org/record/830025#.Wcg29Fd2O1E" } ] }, { "id": "20540", "date": "31 Mar 2017", "name": "Jeremy N. Harvey", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe very short manuscript reports homology modelling of the membrane protein, coupled with 'validation' by using vertical excitation energies computed with a QM/MM protocol. I think the work is interesting, particularly since the authors have made their data available, making it easier to others to build upon it. However, I have a number of concerns, many of which could be addressed simply by describing the results and their implications in a bit more detail.\nThe first set of comments (1)-(3) in Cohen & Farhi's report seem important to me. I think that in the paper, the authors use the position of the absorption peak to judge the validity of their structural model. So the precise mechanism of fluorescence does not matter, nor does proton transfer, nor does the fluorescence quantum yield. Rather these are ideas for future work. But it would do no harm to spell these things out a bit more.\n\nThere is very little in the results section: 1 Figure, 1 Table, and 4 lines of text. Why not tell us a bit more? For example, the authors generate a homology model, but apart from showing us a cartoon structural picture of it, they don't say anything about it. How good was the sequence alignment? How long are the various helices? How well does this compare to various other transmembrane proteins of this type? How much did the other homology models differ from the preferred one, and from each other? Without this kind of analysis, this paper really just consists of a set of input and output files of homology modelling/sequence alignment.\n\nStill in the results section, the authors use SORCI+Q QM/MM vertical excitation energies computed at optimized structures to assess the quality of the homology models. This seems extremely naive to me. The different models return vertical excitation energies that differ by just over 1%, and are about 4% from experiment. I really don't think that this is a good basis for deciding that one of them is better than the others. There are just too many sources of error for making this a meaningful comparison. On the one hand, SORCI+Q with the basis set used will potentially have an error of several tenths of an eV. 0.2 eV is 10% of the excitation energy based on the experimental spectrum. Furthermore, the authors have computed just one vertical excitation energy for each model, at a structure derived by a rather minimal geometry optimization ('relaxation') protocol. The FWHM of the UV/Vis absorption peak of bacetriorhodopsin appears to be about 100nm, i.e. 15% of the band centre. I assume roughly the same is true here. In principle, even accepting that the starting point homology model is correct, and the SORCI Hamiltonian is exact, the QM/MM protocol used could return a vertical excitation energy 50 nm or so either side of the experimental peak. This just makes the criterion meaningless. There are other criteria for assessing the internal consistency of homology models.\n\nAs I understand it, the philosophy of F1000Research is that papers can be published if they have some interest, and I think some people might find the homology modelling interesting, and the detailed structural models with the chromophore. So I'm happy for this to be indexed. But I think the QM/MM calculations are hugely over-interpreted, and recommend the authors consider revising this section, as well as adding some of the discussion I mention above.", "responses": [ { "c_id": "3177", "date": "15 Nov 2017", "name": "Mikhail N. Ryazantsev", "role": "Author Response", "response": "Dear Prof. Harvey, Thank you for your review of our article “A voltage-dependent fluorescent indicator for optogenetic applications, archaerhodopsin-3: Structure and optical properties from in silico modeling”. We have carefully read it and made several changes of the article, considering your notices. We attach the new version of the article in this email. 1. “There is very little in the results section: 1 Figure, 1 Table, and 4 lines of text. Why not tell us a bit more? For example, the authors generate a homology model, but apart from showing us a cartoon structural picture of it, they don't say anything about it. How good was the sequence alignment? How long are the various helices? How well does this compare to various other transmembrane proteins of this type? How much did the other homology models differ from the preferred one, and from each other? Without this kind of analysis, this paper really just consists of a set of input and output files of homology modelling/sequence alignment.”Considering this remark, we edited the results section of our article. The sequence identity between Arch-1 and Arch-3 is 84.5%, sequence similarity is 91.5% (based on the AlignMe alignment of these sequences). We added this information in the “Methods” section of our article.We also compared the results of the sequence alignment algorithms. The comparison showed that all three of them are very similar: AlignMe and MUSTER gave identical results with small difference from the MP-T alignment in the beginning of the sequence (Figure 1 in the article). We added the following text in the “Results” section of our article: “On the first step of structure prediction of archaerhodopsin-3 we performed the alignment of its amino acid sequence with the sequence of archaerhodopsin-1, comparing three different algorithms: AlignMe, MUSTER and MP-T. The results of AlignMe and MUSTER algorithms were identical; they differ from the MP-T alignment result in the flexible N-terminal part (Figure 1, a-c).”We also compared the crystallographic structure of archaerhodopsin-1 and the predicted model of archaerhodopsin-3 (Figure 2, 3 in the article). We showed that the structures of two proteins are very similar, and they differ only in loop regions and on the edges of alpha-helices, i.e. in the regions located quite far from the chromophore. We added the following text in the “Results” section of our article:“Comparison of crystallographic structure of archaerhodopsin-1 with the predicted structure of archaerhodopsin-3 showed that these two proteins differ only in loop regions and on the edges of the alpha-helices (Figure 2, 3). These regions are located on relatively large distance from the retinal chromophore.” 2. “Still in the results section, the authors use SORCI+Q QM/MM vertical excitation energies computed at optimized structures to assess the quality of the homology models. This seems extremely naive to me. The different models return vertical excitation energies that differ by just over 1%, and are about 4% from experiment. I really don't think that this is a good basis for deciding that one of them is better than the others. There are just too many sources of error for making this a meaningful comparison. On the one hand, SORCI+Q with the basis set used will potentially have an error of several tenths of an eV. 0.2 eV is 10% of the excitation energy based on the experimental spectrum. Furthermore, the authors have computed just one vertical excitation energy for each model, at a structure derived by a rather minimal geometry optimization ('relaxation') protocol. The FWHM of the UV/Vis absorption peak of bacetriorhodopsin appears to be about 100nm, i.e. 15% of the band centre. I assume roughly the same is true here. In principle, even accepting that the starting point homology model is correct, and the SORCI Hamiltonian is exact, the QM/MM protocol used could return a vertical excitation energy 50 nm or so either side of the experimental peak. This just makes the criterion meaningless. There are other criteria for assessing the internal consistency of homology models.”To refer to these comments, we modified the text. In this case, all models provide reasonable results. However, it is not always the point, and predicted structures can give easily more than 50 nm off experimental values after a relatively small change of three-dimensional structure. In other words, modeling of absorption maxima is very sensitive to the quality of three-dimensional structures.Considering these remarks, we added the following modifications in the “Results” section:“All obtained models provide reasonable results – the deviation range is only 31 nm from the experiment (Table 1). This deviation range is sensible considering the approximations used in our methodologies. In our previous studies, we obtained a 29 nm shift for the absorption maximum of halorhodopsin from N.pharaonis, a 25 nm shift for the absorption maximum of archaerhodopsin-2 and a 39 nm shift for the channelrhodopsin-2.”" } ] } ]	2	https://f1000research.com/articles/6-33
https://f1000research.com/articles/6-2010/v1	14 Nov 17	{ "type": "Software Tool Article", "title": "Easy and efficient ensemble gene set testing with EGSEA", "authors": [ "Monther Alhamdoosh", "Charity W. Law", "Luyi Tian", "Julie M. Sheridan", "Milica Ng", "Matthew E. Ritchie", "Charity W. Law", "Luyi Tian", "Julie M. Sheridan", "Milica Ng" ], "abstract": "Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.", "keywords": [ "gene expression", "RNA-sequencing", "microarrays", "gene set enrichment", "Bioconductor" ], "content": "Introduction\n\nGene set enrichment analysis allows researchers to efficiently extract biological insights from long lists of differentially expressed genes by interrogating them at a systems level. In recent years, there has been a proliferation of gene set enrichment (GSE) analysis methods released through the Bioconductor project1 together with a steady increase in the number of gene set collections available through online databases such as MSigDB2, GeneSetDB3 and KEGG4. In an effort to unify these computational methods and knowledge-bases, the EGSEA R/Bioconductor package was developed. EGSEA, which stands for Ensemble of Gene Set Enrichment Analyses5 combines the results from multiple algorithms to arrive at a consensus gene set ranking to identify biological themes and pathways perturbed in an experiment. EGSEA calculates seven statistics to combine the individual gene set statistics of base GSE methods to rank biologically relevant gene sets. The current version of the EGSEA package6 utilizes the analysis results of up to twelve prominent GSE algorithms that include: ora7, globaltest8, plage9, safe10, zscore11, gage12, ssgsea13, padog14, gsva15, camera16, roast17 and fry17. The ora, gage, camera and gsva methods depend on a competitive null hypothesis which assumes the genes in a set do not have a stronger association with the experimental condition compared to randomly chosen genes outside the set. The remaining eight methods are based on a self-contained null hypothesis that only considers genes within a set and again assumes that they have no association with the experimental condition.\n\nEGSEA provides access to a diverse range of gene signature collections through the EGSEAdata package that includes more than 25,000 gene sets for human and mouse organised according to their database sources (Table 1). For example, MSigDB2 includes a number of collections (Hallmark (h) and c1–c7) that explore different biological themes ranging from very broad (h, c2, c5) through to more specialised ones focusing on cancer (c4, c6) and immunology (c7). The other main sources are GeneSetDB3 and KEGG4 which have similar collections focusing on different biological characteristics (Table 1). The choice of collection/s in any given analysis should of course be guided by the biological question of interest. The MSigDB c2 and c5 collections are the most widely used in our own analysis practice, spanning a wide range of biological processes and can often reveal new biological insights when applied to a given dataset.\n\nThe purpose of this article is to demonstrate the gene set testing workflow available in EGSEA on both RNA-seq and microarray data. Each analysis involves four major steps that are summarized in Figure 1: (1) selecting appropriate gene set collections for analysis and building an index that maps between the members of each set and the expression matrix; (2) choosing the base GSE methods to combine and the ranking options; (3) running the EGSEA test and (4) reporting results in various ways to share with collaborators. The EGSEA functions involved in each of these steps are introduced with code examples to demonstrate how they can be deployed as part of a limma differential expression analysis to help with the interpretation of results.\n\n\nGene expression profiling of the mouse mammary gland\n\nThe first experiment analysed in this workflow is an RNA-seq dataset from Sheridan et al. (2015)18 that consists of 3 cell populations (Basal, Luminal Progenitor (LP) and Mature Luminal (ML)) sorted from the mammary glands of female virgin mice. Triplicate RNA samples from each population were obtained in 3 batches and sequenced on an Illumina HiSeq 2000 using a 100 base-pair single-ended protocol. Raw sequence reads from the fastq files were aligned to the mouse reference genome (mm10) using the Rsubread package19. Next, gene-level counts were obtained using featureCounts20 based on Rsubread’s built-in mm10 RefSeq-based annotation. The raw data along with further information on experimental design and sample preparation can be downloaded from the Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo/) using GEO Series accession number GSE63310 and will be preprocessed according to the RNA-seq workflow published by Law et al. (2016)21.\n\nThe second experiment analysed in this workflow comes from Lim et al. (2010)22 and is the microarray equivalent of the RNA-seq dataset mentioned above. The same 3 populations (Basal (also referred to as “MaSC-enriched”), LP and ML) were sorted from mouse mammary glands via flow cytometry. Total RNA from 5 replicates of each cell population were hybridised onto 3 Illumina MouseWG-6 v2 BeadChips. The intensity files and chip annotation file available in Illumina’s proprietary formats (IDAT and BGX respectively) can be downloaded from http://bioinf.wehi.edu.au/EGSEA/arraydata.zip. The raw data from this experiment is also available from GEO under Series accession number GSE19446.\n\n\nAnalysis of RNA-seq data with EGSEA\n\nOur RNA-seq analysis follows on directly from the workflow of Law et al. (2016) which performs a differential gene expression analysis on this data set using the Bioconductor packages edgeR23, limma24 and Glimma25 with gene annotation from the Mus.musculus package26. The limma package offers a well-developed suite of statistical methods for dealing with differential expression for both microarray and RNA-seq datasets and will be used in the analyses of both datasets presented in this workflow.\n\nTo get started with this analysis, download the R data file from http://bioinf.wehi.edu.au/EGSEA/mam.rnaseq.rdata. The code below loads the preprocessed count matrix from Law et al. (2016), performs TMM normalisation27 on the raw counts, and calculates voom weights for use in comparisons of gene expression between Basal and LP, Basal and ML, and LP and ML populations.\n\n\n\nThe voom function28 from the limma package converts counts to log-counts-per-million (log-cpm) and calculates observation-level precision weights. The voom object (v) contains normalized log-cpm values and gene information used by all of the methods in the EGSEA analysis below. The precision weights stored within v are also used by the camera, roast and fry gene set testing methods.\n\n\n\nFor further information on preprocessing see Law et al. (2016), as a detailed explanation of these steps is beyond the scope of this article.\n\n\nGene set testing\n\nThe EGSEA algorithm makes use of the voom object (v), a design matrix (design) and an optional contrasts matrix (contr.matrix). The design matrix describes how the samples in the experiment relate to the coefficients estimated by the linear model29. The contrasts matrix then compares two or more of these coefficients to allow relative assessment of differential expression. Base methods that utilize linear models such as those from limma and GSVA (gsva, plage, zscore and ssgsea) make use of the design and contrasts matrices directly. For methods that do not support linear models, these two matrices are used to extract the group information for each comparison.\n\nThe package EGSEAdata includes more than 25,000 gene sets organized in collections depending on their database sources. Summary information about the gene set collections available in EGSEAdata can be displayed as follows:\n\n\n\nAs the output above suggests, users can obtain help on any of the collections using the standard R help (?) command, for instance ?Mm.c2 will return more information on the mouse version of the c2 collection from MSigDB. The above information can be returned as a list:\n\n\n\nTo highlight the capabilities of the EGSEA package, the KEGG pathways, c2 (curated gene sets) and c5 (Gene Ontology gene sets) collections from the MSigDB database are selected. Next, an index is built for each gene set collection using the EGSEA indexing functions to link the genes in the different gene set collections to the rows of our RNA-seq gene expression matrix. Indexes for the c2 and c5 collections from MSigDB and for the KEGG pathways are built using the buildIdx function which relies on Entrez gene IDs as its key. In the EGSEAdata gene set collections, Entrez IDs are used as they are widely adopted by the different source databases and tend to be more consistent and robust since there is one identifier per gene in a gene set. It is also relatively easy to convert other gene IDs into Entrez IDs.\n\n\n\nTo obtain additional information on the gene set collection indexes, including the total number of gene sets, the version number and date of last revision, the methods summary, show and getSetByName (or getSetByID) can be invoked on an object of class GSCollectionIndex, which stores all of the relevant gene set information, as follows:\n\n\n\nObjects of class GSCollectionIndex store for each gene set the Entrez gene IDs in the slot original, the indexes in the slot idx and additional annotation for each set in the slot anno.\n\n\n\nOther EGSEA functions such as buildCustomIdx, buildGMTIdx, buildKEGGIdx, buildMSigDBIdx and buildGeneSetDBIdx can be also used to build gene set collection indexes. The functions buildCustomIdx and buildGMTIdx were written to allow users to run EGSEA on gene set collections that may have been curated within a lab or downloaded from public databases and allow use of gene identifiers other than Entrez IDs. Example databases include, ENCODE Gene Set Hub (available from https://sourceforge.net/projects/encodegenesethub/), which is a growing resource of gene sets derived from high quality ENCODE profiling experiments encompassing hundreds of DNase hypersensitivity, histone modification and transcription factor binding experiments30. Other resources include PathwayCommons (http://www.pathwaycommons.org/)31 and the KEGGREST32 package that provides access to up-to-date KEGG pathways across many species.\n\nBefore an EGSEA test is carried out, a few parameters need to be specified. First, a mapping between Entrez IDs and Gene Symbols is created for use by the visualization procedures. This mapping can be extracted from the genes data.frame of the voom object as follows:\n\n\n\nAnother important parameter in EGSEA is the list of base GSE methods (baseMethods in the code below), which determines the individual algorithms that are used in the ensemble testing. The supported base methods can be listed using the function egsea.base as follows:\n\n\n\nThe plage, zscore and ssgsea algorithms are available in the GSVA package and camera, fry and roast are implemented in the limma package24. The ora method is implemented using the phyper function from the stats package33, which estimates the hypergeometric distribution for a 2 × 2 contingency table. The remaining algorithms are implemented in Bioconductor packages of the same name. A wrapper function is provided for each individual GSE method to utilize this existing R code and create a universal interface for all methods.\n\nEleven base methods are selected for our EGSEA analysis: camera, safe, gage, padog, plage, zscore, gsva, ssgsea, globaltest, ora and fry. Fry is a fast approximation of roast that assumes equal gene-wise variances across samples to produce similar p-values to a roast analysis run with an infinite number of rotations, and is selected here to save time.\n\n\n\nAlthough, different combinations of base methods might produce different results, it has been found via simulation that including more methods gives better performance5.\n\nSince each base method generates different p-values, EGSEA supports six different methods from the metap package34 for combining individual p-values (Wilkinson35 is default), which can be listed as follows:\n\n\n\nFinally, the sorting of EGSEA results plays an essential role in identifying relevant gene sets. Any of EGSEA’s combined scores or the rankings from individual base methods can be used for sorting the results.\n\n\n\nAlthough p.adj is the default option for sorting EGSEA results for convenience, we recommend the use of either med.rank or vote.rank because they efficiently utilize the rankings of individual methods and tend to produce fewer false positives5.\n\nNext, the EGSEA analysis is performed using the egsea function that takes a voom object, a contrasts matrix, collections of gene sets and other run parameters as follows:\n\n\n\nIn situations where the design matrix includes an intercept, a vector of integers that specify the columns of the design matrix to test using EGSEA can be passed to the contrasts argument. If this parameter is NULL, all pairwise comparisons based on v$targets$group are created, assuming that group is the primary factor in the design matrix. Likewise, all the coefficients of the primary factor are used if the design matrix has an intercept.\n\nEGSEA is implemented with parallel computing features enabled using the parallel package33 at both the method-level and experimental contrast-level. The running time of the EGSEA test depends on the base methods selected and whether report generation is enabled or not. The latter significantly increases the run time, particularly if the argument display.top is assigned a large value (> 20) and/or a large number of gene set collections are selected. EGSEA reporting functionality generates set-level plots for the top gene sets as well as collection-level plots.\n\nThe EGSEA package also has a function named egsea.cnt, that can perform the EGSEA test using an RNA-seq count matrix rather than a voom object, a function named egsea.ora, that can perform over-representation analysis with EGSEA reporting capabilities using only a vector of gene IDs, and the egsea.ma function that can perform EGSEA testing using a microarray expression matrix as shown later in the workflow.\n\nClasses used to manage the results. The output of the functions egsea, egsea.cnt, egsea.ora and egsea.ma is an S4 object of class EGSEAResults. Several S4 methods can be invoked to query this object. For example, an overview of the EGSEA analysis can be displayed using the show method as follows:\n\n\n\nThis command displays the number of genes and samples that were included in the analysis, the experimental contrasts, base GSE methods, the method used to combine the p-values derived from different GSE algorithms, the sorting statistic used and the size of each gene set collection. Note that the gene set collections are identified using the labels that appear in parentheses (e.g. c2) in the output of show.\n\nGetting top ranked gene sets. A summary of the top 10 gene sets in each collection for each contrast in addition to the EGSEA comparative analysis can be displayed using the S4 method summary as follows:\n\n\n\nEGSEA’s comparative analysis allows researchers to estimate the significance of a gene set across multiple experimental contrasts. This analysis helps in the identification of biological processes that are perturbed in multiple experimental conditions simultaneously. This experiment is the RNA-seq equivalent of Lim et al. (2010)22, who used Illumina microarrays to study the same cell populations (see later), so it is reassuring to observe the LIM gene signatures derived from this experiment amongst the top ranked c2 gene signatures in both the individual contrasts and comparative results.\n\nAnother way of exploring the EGSEA results is to retrieve the top ranked N sets in each collection and contrast using the method topSets. For example, the top 10 gene sets in the c2 collection for the comparative analysis can be retrieved as follows:\n\n\n\nThe gene sets are ordered based on their med.rank as selected when egsea was invoked above. When the argument names.only is set to FALSE, additional information is displayed for each gene set including gene set annotation, the EGSEA scores and the individual rankings by each base method. As expected, gene sets retrieved by EGSEA included the LIM gene sets22 that were derived from microarray profiles of analagous mammary cell populations (sets 1, 2, 4, 6 and 8) as well as those derived from populations with similar origin (sets 7 and 9) and behaviour or characteristics (sets 5 and 10).\n\nNext, topSets can be used to search for gene sets of interest based on different EGSEA scores as well as the rankings of individual methods. For example, the ranking of the six LIM gene sets from the c2 collection can be displayed based on the med.rank as follows:\n\n\n\nWhile five of the LIM gene sets are ranked in the top 10 by EGSEA, the values shown in the median rank (med.rank) column indicate that individual methods can assign much lower ranks to these sets. EGSEA’s prioritisation of these gene sets demonstrates the benefit of an ensemble approach.\n\nSimilarly, we can find the top 10 pathways in the KEGG collection from the ensemble analysis for the Basal versus LP contrast and the comparative analysis as follows:\n\n\n\nEGSEA highlights many pathways with known importance in the mammary gland such as those associated with distinct roles in lactation like basal cell contraction (Vascular smooth muscle contraction and Oxytocin signalling pathway) and milk production and secretion from luminal lineage cells (Collecting duct acid secretion, Synaptic vesicle cycle and Lysosome).\n\nVisualizing results at the gene set level. Graphical representation of gene expression patterns within and between gene sets is an essential part of communicating the results of an analysis to collaborators and other researchers. EGSEA enables users to explore the elements of a gene set via a heatmap using the plotHeatmap method. Figure 2 shows examples for the LIM_MAMMARY_STEM_CELL_UP and LIM_MAMMARY_STEM_CELL_DN signatures which can be visualized across all contrasts using the code below.\n\n\n\nWhen using plotHeatmap, the gene.set value must match the name returned from the topSets method. The rows of the heatmap represent the genes in the set and the columns represent the experimental contrasts. The heatmap colour-scale ranges from down-regulated (blue) to up-regulated (red) while the row labels (Gene symbols) are coloured in green when the genes are statistically significant in the DE analysis (i.e. FDR ≤ 0.05 in at least one contrast). Heatmaps can be generated for individual comparisons by changing the contrast argument of plotHeatmap. The plotHeatmap method also generates a CSV file that includes the DE analysis results from limma::topTable for all expressed genes in the selected gene set and for each contrast (in the case of contrast = \"comparison\"). This file can be used to create customised plots using other R/Bioconductor packages.\n\nIn addition to heatmaps, pathway maps can be generated for the KEGG gene sets using the plotPathway method which uses functionality from the pathview package36. For example, the third KEGG signalling pathway retrieved for the contrast BasalvsLP is Vascular smooth muscle contraction and can be visualized as follows:\n\n\n\nPathway components are coloured based on the gene-specific log-fold-changes as calculated in the limma DE analysis (Figure 3). Similarly, a comparative map can be generated for a given pathway across all contrasts.\n\n\n\nThe comparative pathway map shows the log-fold-changes for each gene in each contrast by dividing the gene nodes on the map into multiple columns, one for each contrast (Figure 4).\n\nVisualizing results at the experiment level. Since EGSEA combines the results from multiple gene set testing methods, it can be interesting to compare how different base methods rank a given gene set collection for a selected contrast. The plotMethods command generates a multi-dimensional scaling (MDS) plot for the ranking of gene sets across all the base methods used (Figure 5). Methods that rank gene sets similarly will appear closer together in this plot and we see that certain methods consistently cluster together across different gene set collections. The clustering of methods does not necessarily follow the style of null hypothesis tested though (i.e. self-contained versus competitive).\n\n\n\nMulti-dimensional scaling (MDS) plot showing the relationship between different gene set testing methods based on the rankings of the c2 (a) and c5 (b) gene sets on the Basal vs LP contrast.\n\nThe significance of each gene set in a given collection for a selected contrast can be visualized using EGSEA’s plotSummary method.\n\n\n\nThe summary plot visualizes the gene sets as bubbles based on the − log10 (p-value) (X-axis) and the average absolute log fold-change of the set genes (Y-axis). The sets that appear towards the top-right corner of this plot are most likely to be biologically relevant. EGSEA generates two types of summary plots: the directional summary plot (Figure 6a), which colours the bubbles based on the regulation direction of the gene set (the direction of the majority of genes), and the ranking summary plot (Figure 6b), which colours the bubbles based on the gene set ranking in a given collection (according to the sort.by argument). The bubble size is based on the EGSEA significance score in the former plot and the gene set size in the latter. For example, the summary plots of the KEGG pathways for the LP vs ML contrast show few significant pathways (Figure 6). The blue colour labels on the ranking plot represents gene sets that do not appear in the top 10 gene sets that are selected based on the sort.by argument, yet their EGSEA significance scores are among the top 5 in the entire collection based on the significance score. This is used to identify gene sets with high significance scores that were not captured by the sort.by score. The gene set IDs and more information about each set can be found in the EGSEA HTML report generated later.\n\nBy default, plotSummary uses a gene set’s p.adj score for the X-axis. This behaviour can be easily modified by assigning any of the available sort.by scores into the parameter x.axis, for example, med.rank can be used to create an EGSEA summary plot (Figure 7a) as follows:\n\n\n\nThe x-axis in each plot is the med.rank. A cut-off of 300 was used to select significant gene sets in the filtered plot (b).\n\nThe summary plot tends to become cluttered when the size of the gene set collection is very large as in Figure 7a. The parameter x.cutoff can be used to focus in on the significant gene sets rather than plotting the entire gene set collection, for example (Figure 7b):\n\n\n\nComparative summary plots can be also generated to compare the significance of gene sets between two contrasts, for example, the comparison between Basal vs LP and Basal vs ML (Figure 8a) shows that most of the KEGG pathways are regulated in the same direction with relatively few pathways regulated in opposite directions (purple coloured bubbles in Figure 8a). Such figures can be generated using the plotSummary method as follows:\n\n\n\nThe plotSummary method has two useful parameters: (i) use.names that can be used to display gene set names instead of gene set IDs and (ii) interactive that can be used to generate an interactive version of this plot.\n\nThe c5 collection of MSigDB and the Gene Ontology collection of GeneSetDB contain Gene Ontology (GO) terms. These collections are meant to be non-redundant, containing only a small subset of the entire GO and visualizing how these terms are related to each other can be informative. EGSEA utilizes functionality from the topGO package37 to generate GO graphs for the significant biological processes (BPs), cellular compartments (CCs) and molecular functions (MFs). The plotGOGraph method can generate such a display (Figure 9) as follows:\n\n\n\nThe GO graphs are coloured based on the values of the argument sort.by, which in this instance was taken as med.rank by default since this was selected when EGSEA was invoked. The top five most significant GO terms are highlighted by default in each GO category (MF, CC or BP). More terms can be displayed by changing the value of the parameter noSig. However, this might generate very complicated and unresolved graphs. The colour of the nodes varies between red (most significant) and yellow (least significant). The values of the sort.by scoring function are scaled between 0 and 1 to generate these graphs.\n\nAnother way to visualize results at the experiment level is via a summary bar plot. The method plotBars can be used to generate a bar plot for the top N gene sets in an individual collection for a particular contrast or from a comparative analysis across multiple contrasts. For example, the top 20 gene sets of the comparative analysis carried out on the c2 collection of MSigDB can be visualized in a bar plot (Figure 10) as follows:\n\n\n\nThe colour of the bars is based on the regulation direction of the gene sets, i.e., red for up-regulated, blue for down-regulated and purple for neutral regulation (in the case of the comparative analysis on experimental contrasts that show opposite behaviours). By default, the − log10(p.ad j) values are plotted for the top 20 gene sets selected and ordered based on the sort.by parameter. The parameters bar.vals, number and sort.by of plotBars can be changed to customize the bar plot.\n\nWhen changes over multiple conditions are of interest, a summary heatmap can be a useful visualization. The method plotSummaryHeatmaps generates a heatmap of the top N gene sets in the comparative analysis across all experimental conditions (Figure 11). By default, 20 gene sets are selected based on the sort.by parameter and the values plotted are the average log-fold changes at the set level for the genes regulated in the same direction as the set regulation direction, i.e. avg.logfc.dir. The parameters number, sort.by and hm.vals of the plotSummaryHeatmaps can be used to customize the summary heatmap. Additionally, the parameter show.vals can be used to display the values of a specific EGSEA score on the heatmap cells. An example summary heatmap can be generated for the MSigDB c2 collection with the following code:\n\n\n\nSummary heatmaps for the top 20 gene sets from the c2 (a) and KEGG (b) collections obtained from the EGSEA comparative analysis.\n\nWe find the heatmap view at both the gene set and summary level and the summary level bar plots to be useful summaries to include in publications to highlight the gene set testing results. The top differentially expressed genes from each contrast can be accessed from the EGSEAResults object using the limmaTopTable method.\n\n\n\nCreating an HTML report of the results. To generate an EGSEA HTML report for this dataset, you can either set report=TRUE when you invoke egsea or use the S4 method generateReport as follows:\n\n\n\nThe EGSEA report generated for this dataset is available online at http://bioinf.wehi.edu.au/EGSEA/mam-rnaseq-egsea-report/index.html (Figure 12). The HTML report is a convenient means of organising all of the results generated up to now, from the individual tables to the gene set level heatmaps, pathway maps and summary level plots. It can easily be shared with collaborators to allow them to explore their results more fully. Interactive tables of results via the DT package (https://CRAN.R-project.org/package=DT) and summary plots from plotly (https://CRAN.R-project.org/package=plotly) are integrated into the report using htmlwidgets (https://CRAN.R-project.org/package=htmlwidgets) and can be added by setting interactive = TRUE in the command above. This option significantly increases both the run time and size of the final report due to the large number of gene sets in most collections.\n\nThis summary page details the analysis parameters (methods combined and ranking options selected) and organises the gene set analysis results by contrast, with further separation by gene set collection. The final section on this page presents results from the comparative analysis. For each contrast and gene set collection analysed, links to tables of results and plots are provided.\n\nThis example completes our overview of EGSEA’s gene set testing and plotting capabilities for RNA-seq data. Readers can refer to the EGSEA vignette or individual help pages for further details on each of the above methods and classes.\n\n\nAnalysis of microarray data with EGSEA\n\nThe second dataset analysed in this workflow comes from Lim et al. (2010)22 and is the microarray equivalent of the RNA-seq data analysed above. Support for microarray data is a new feature in EGSEA, and in this example, we show an express route for analysis according to the steps shown in Figure 1, from selecting gene sets and building indexes, to configuring EGSEA, testing and reporting the results. First, the data must be appropriately preprocessed for an EGSEA analysis and to do this we make use of functions available in limma.\n\n\nReading, preprocessing and normalisation of microarray data\n\nTo analyse this dataset, we begin by unzipping the files downloaded from http://bioinf.wehi.edu.au/EGSEA/arraydata.zip into the current working directory. Illumina BeadArray data can be read in directly using the readIDAT and readBGX functions from the illuminaio package38. However, a more convenient way is via the read.idat function in limma which uses these illuminaio functions and outputs the data as an EListRaw object for further processing.\n\n\n\nNext the neqc function in limma is used to carry out normexp background correction and quantile normalisation on the raw intensity values using negative control probes39. This is followed by log2-transformation of the normalised intensity values and removal of the control probes.\n\n\n\nWe then filter out probes that are consistently non-expressed or lowly expressed throughout all samples as they are uninformative in downstream analysis. Our threshold for expression requires probes to have a detection p-value of less than 0.05 in at least 5 samples (the number of samples within each group). We next remove genes without a valid Entrez ID and in cases where there are multiple probes targeting different isoforms of the same gene, select the probe with highest average expression as the representative one to use in the EGSEA analysis. This leaves 7,123 probes for further analysis.\n\n\n\n\nSetting up the linear model for EGSEA testing\n\nAs before, we need to set up an appropriate linear model29 and contrasts matrix to look for differences between the Basal and LP, Basal and ML and LP and ML populations. A batch term is included in the linear model to account for differences in expression that are attributable to the day the experiment was run.\n\n\n\n\n1. Creating gene set collection indexes\n\nWe next extract the mouse c2, c5 and KEGG gene signature collections from the EGSEAdata package and build indexes based on Entrez IDs that link between the genes in each signature and the rows of our expression matrix.\n\n\n\n\n2. Configuring and 3. Testing with EGSEA\n\nThe same 11 base methods used previously in the RNA-seq analysis were selected for the ensemble testing of the microarray data using the function egsea.ma. Gene sets were again prioritised by their median rank across the 11 methods.\n\n\n\n\n4. Reporting EGSEA results\n\nAn HTML report that includes each of the gene set level and summary level plots shown individually for the RNA-seq analysis was then created using the generateReport function. We complete our analysis by displaying the top ranked sets for the c2 collection from a comparative analysis across all contrasts.\n\n\n\nThe EGSEA report generated for this dataset is available online at http://bioinf.wehi.edu.au/EGSEA/mam-ma-egsea-report/index.html. Reanalysis of this data retrieves similar c2 gene sets to those identified by analysis of RNA-seq data. These included the LIM gene signatures (sets 1, 2 and 3) as well as those derived from populations with similar cellular origin (set 4).\n\n\nDiscussion\n\nIn this workflow article, we have demonstrated how to use the EGSEA package to combine the results obtained from different gene signature databases across multiple GSE methods to find an ensemble solution. A key benefit of an EGSEA analysis is the detailed and comprehensive HTML report that can be shared with collaborators to help them interpret their data. This report includes tables prioritising gene signatures according to the user specified analysis options, and both gene set specific and summary graphics, each of which can be generated individually using specific R commands. The approach taken by EGSEA is facilitated by the diverse range of gene set testing algorithms and plotting capabilities available within Bioconductor. EGSEA has been tailored to suit a limma-based differential expression analysis which continues to be a very popular and flexible platform for transcriptomic data. Analysts who choose an individual GSE algorithm to prioritise their results rather than an ensemble solution can still benefit from EGSEA’s comprehensive reporting capability.\n\n\nSoftware availability\n\nCode to perform this analysis can be found in the EGSEA123 workflow package available from Bioconductor: https://www.bioconductor.org/help/workflows/EGSEA123.\n\nLatest source code is available at: https://github.com/mritchie/EGSEA123.\n\nArchived source code as at the time of publication is available at: https://doi.org/10.5281/zenodo.104343640.\n\nSoftware license: Artistic License 2.0.", "appendix": "Competing interests\n\n\n\nMA and MN are employees of CSL Limited.\n\n\nGrant information\n\nThis work was funded by a National Health and Medical Research Council (NHMRC) Fellowship to MER (GNT1104924), Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS.\n\n\nAcknowledgements\n\nThis material was first trialled in a workshop at the BioC 2017 conference at the Dana Farber Cancer Institute (Boston, MA) on 28 July 2017. We thank the participants at this workshop for their feedback. The authors also thank Dr Alexandra Garnham (The Walter and Eliza Hall Institute of Medical Research) for feedback on this workflow article.\n\n\nReferences\n\nHuber W, Carey VJ, Gentleman R, et al.: Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015; 12(2): 115–21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSubramanian A, Tamayo P, Mootha VK, et al.: Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005; 102(43): 15545–50. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAraki H, Knapp C, Tsai P, et al.: GeneSetDB: A comprehensive meta-database, statistical and visualisation framework for gene set analysis. FEBS Open Bio. 2012; 2: 76–82. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nKanehisa M, Goto S: KEGG: kyoto encyclopedia of genes and genomes. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSheridan JM, Ritchie ME, Best SA, et al.: A pooled shRNA screen for regulators of primary mammary stem and progenitor cells identifies roles for Asap1 and Prox1. BMC Cancer. 2015; 15(1): 221. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiao Y, Smyth GK, Shi W: The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013; 41(10): e108. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLiao Y, Smyth GK, Shi W: featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014; 30(7): 923–30. PubMed Abstract \| Publisher Full Text\n\nLaw CW, Alhamdoosh M, Su S, et al.: RNA-seq analysis is easy as 1-2-3 with limma, Glimma and edgeR [version 2; referees: 3 approved]. F1000Res. 2016; 5: 1408. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLim E, Wu D, Pal B, et al.: Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways. Breast Cancer Res. 2010; 12(2): R21. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRobinson MD, McCarthy DJ, Smyth GK: edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010; 26(1): 139–40. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nRitchie ME, Phipson B, Wu D, et al.: limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015; 43(7): e47. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSu S, Law CW, Ah-Cann C, et al.: Glimma: interactive graphics for gene expression analysis. Bioinformatics. 2017; 33(13): 2050–2. PubMed Abstract \| Publisher Full Text\n\nBioconductor Core Team: Mus.musculus: Annotation package for the Mus.musculus object. R package version 1.3.1. 2015. Publisher Full Text\n\nRobinson MD, Oshlack A: A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010; 11(3): R25. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nLaw CW, Chen Y, Shi W, et al.: Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol. 2014; 15(2): R29. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nSmyth GK: Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol. 2004; 3(1): Article 3. PubMed Abstract \| Publisher Full Text\n\nZiemann M, Kaspi A, Rafehi H, et al.: The ENCODE Gene Set Hub. Lorne Genome Conference. 2017. Publisher Full Text\n\nCerami EG, Gross BE, Demir E, et al.: Pathway commons, a web resource for biological pathway data. Nucleic Acids Res. 2011; 39(Database issue): D685–D690. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nTenenbaum D: KEGGREST: Client-side REST access to KEGG. R package version 1.16.0. 2017.\n\nR Core Team: R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, 2017. Reference Source\n\nDewey M: metap: meta-analysis of significance values. R package version 0.8. 2017. Reference Source\n\nWilkinson B: A statistical consideration in psychological research. Psychol Bull. 1951; 48(3): 156–8. PubMed Abstract \| Publisher Full Text\n\nLuo W, Brouwer C: Pathview: an R/Bioconductor package for pathway-based data integration and visualization. Bioinformatics. 2013; 29(14): 1830–1. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nAlexa A, Rahnenfuhrer J: topGO: Enrichment Analysis for Gene Ontology. R package version. 2016. Publisher Full Text\n\nSmith ML, Baggerly KA, Bengtsson H, et al.: illuminaio: an open source idat parsing tool for Illumina microarrays [version 1; referees: 2 approved]. F1000Res. 2013; 2: 264. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nShi W, Oshlack A, Smyth GK: Optimizing the noise versus bias trade-off for Illumina Whole Genome Expression Beadchips. Nucleic Acids Res. 2010; 38(22): e204. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nmritchie: mritchie/EGSEA123: F1000 Research article version 1 (Version v1). Zenodo. 2017. Data Source" }	[ { "id": "27972", "date": "27 Nov 2017", "name": "Robert Castelo", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis article describes a gene set enrichment analysis (GSEA) workflow for the \"Ensembl of GSEA\" (EGSEA) R/Bioconductor software package. EGSEA is an ensemble-like method recently published1 by the authors of this workflow that allows the user to simultaneously apply different GSEA algorithms on a high-throughput molecular profiling data set, by combining p-values associated with each algorithm using classical meta-analysis approaches such as the Fisher's method.\nBecause the statistical methodology is already described in detail in the corresponding publication, the present software tool article focuses on showing a step-by-step workflow with EGSEA. However, the vignette of the software package already provides a very detailed description about how to use EGSEA through its 39 pages. Therefore, it would be useful for the interested reader to find upfront when he/she should be consulting the vignette and when he/she should be consulting this workflow. Besides this introductory aspects, the following issues should be addressed before approval:\n\nThe code given in the article breaks, at least in my computer, more concretely, at this line:\ngsa = egsea(voom.results=v, contrasts=contr.matrix,\n\ngs.annots=gs.annots, symbolsMap=symbolsMap,\n\nbaseGSEAs=baseMethods, sort.by=\"med.rank\",\n\nnum.threads = 8, report = FALSE) EGSEA analysis has started ##------ Mon Nov 27 12:37:42 2017 ------## Log fold changes are estimated using limma package ... limma DE analysis is carried out ... Number of used cores has changed to 4 in order to avoid CPU overloading. EGSEA is running on the provided data and c2 collection .......camera....safe...gage.padog....gsva..fry...plage...globaltest...zscore...ora...ssgsea*\n\nError in temp.results[[baseGSEA]][[i]][names(gs.annot@idx), ] :\n\nincorrect number of dimensions\nwhile running it with the latest release version 1.6.0. This is strange since the package builds and runs the vignette without problems. So, this might be related to the different sample data sets. A possible hint may come from the fact that the 'buildIdx()' call is not returning the expected class of object, according to the workflow:\nclass(gs.annots$s2) ## [1] \"NULL\" summary(gs.annots$s2) ## Length Class\n\nMode ##\n\n0\n\nNULL\n\nNULL\n\nThe workflow contains a rather high amount of code, often with a non-trivial use of externally instantiated objects and nested calls to functions. It would be helpful for the interested reader to be able to easily copy and paste the instructions, but the fact that R commands are given with the R shell '>' and '+' symbols makes it less easy. A non-expert user may even copy those characters and get an error. I would recommend removing those characters from the illustrated code, just as it happens with the vignette.\n\nThe workflow assumes that the user has a 'DGEList' object with gene metadata including the mapping between Entrez identifiers' and HGNC symbols. This is a rather unrealistic assumption and I would recommend that the workflow starts building that object from scratch and showing how to build that table of gene metadata.\nBelow I also describe other issues that I would recommend to be considered in future versions of the software but which I do not consider them to be required for approval of this article:\nThe so-called \"summary plot\" shows the -log10 p-value on the x-axis and average absolute log fold-change of the set genes on the y-axis. Because this is in a way analogous to a rotated volcano plot, I would suggest to use the same arrangement of axes as in the volcano plot, which is a rather standardized display of significance and magnitude of the effects of interest.\n\nOne of the key features of the Bioconductor project, to which the EGSEA package is contributing to, is enabling software interoperability through sharing the use of common data structures across different software packages. Using specialized data structures, where analogous ones have been already designed by the Bioconductor core team or by a wider community of developers, locks the user into that package and limits the possibilities of using it as a building block in other more complex workflows. I'm making this comment because I have the impression that the EGSEA package would benefit of using the infrastructure provided by the Bioconductor GSEABase package, in which data structures are defined to store and access gene sets and collections of gene sets of different kinds. A salient feature of that infrastructure is the possibility to seamlessly map gene identifiers of different kinds. This would simplify and improve the user experience of EGSEA since mapping between genes coded with a particular kind of identifier, and gene sets defined with another kind, is one of the most common tasks in a GSEA-like analysis.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "27974", "date": "05 Dec 2017", "name": "Jenny Drnevich", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis F1000 software tool article describes the EGSEA package that incorporates many different gene set testing methods from various packages and also allows access to a wide array of gene sets from different databases through the accompanying EGSEAdata package. These packages will enable researchers to conveniently test many different methods and incorporate their results to get more robust biological insights1, and this article gives a well-written walk-through of how to use the packages.\nThe biggest limitation I see it that EGSEA is focused only on human and mouse data (and rat? The article does not list rat but the help page for buildIdx() lists rat as one of the species). I understand that many of the gene set collections like MSigDB and GeneSetDB are only available for human/mouse, but KEGG currently lists 429 Eukaryotic organisms (http://www.genome.jp/kegg/catalog/org_list.html) and GO terms are readily available for 19 species using BioC's pre-built OrgDB packages and hundreds of other through AnnotationHub. It is unclear whether EGSEA functions buildCustomIdx and buildGMTIdx that were \"written to allow users to run EGSEA on gene set collections that may have been curated within a lab or downloaded from public databases and allow use of gene identifiers other than Entrez IDs\" can be used to run EGSEA on additional species. If so, this should be clearly stated in both the Abstract and in the body of the article, plus an example given on how to use buildCustomIdx for another species. If there is some reason that EGSEA cannot currently extend to other species, this should be acknowledged as a limitation and future versions should strive to allow this (although not required before approval).\n\nOther issues to address before approval:\nI am unable to create the html report on my Windows machine, getting the following error:\nBuild GO DAG topology ..........\nThere are no adj nodes for node: GO:0061857 Error in switch(type, isa = 0, partof = 1, -1) : EXPR must be a length 1 vector\nHowever, I reported this error to the support site (https://support.bioconductor.org/p/103640/#103748) and got a speedy reply from the author. It hopefully will be resolved soon, although there is a concern of why the error was not found on another Windows machine.\n\nI am concerned that as demonstrated in this paper, EGSEA seems to take the place of standard limma differential expression analysis, in that the model fitting takes place within the egsea() function. Certain gene set testing functions do need the individual expression values and not just the fitted values in an MArrayLM object but given the computational time (8 min as shown in the article code block and 19 min on my own computer) you should never run egsea() without first assessing the model fit on your own! Ideally the egsea function could be written to accept MArrayLM, or at least the article should clearly state that users should have first assessed the validity of the model fit through the usual workflow of Law et al. (2016)2 prior to running EGSEA.\n\nI also wonder why there are different interfaces for voom-based analysis and microarray data given that both use EList objects. I understand that the voom weights need to be used internally, but limma's lmFit function handles both without trouble, although it was originally coded for microarray data and the voom functionality came later. Even if there needs to be a separate function egsea.ma() for non-voom, non-count data, it should still accept an EList object so that the user does not have to pull out the expression data and the grouping info.\n\nBack to the computational time required, there are several vague references to removing the roast method \"to save time\" and that the report generation \"significantly\" increases run time. it would be nice to have an example of the time required to run roast and the report generation for the computational architecture that created the article.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Partly\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "27970", "date": "13 Dec 2017", "name": "Weijun Luo", "expertise": [], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nEGSEA is a new gene set analysis tool that combines results from multiple individual tools in R as to yield better results. The authors have published EGSEA methodology previously. This paper focuses on the practical analysis workflow based on EGSEA with specific examples. As EGSEA is a compound and complicated analysis procedure, this work serves as a valuable guidance for the users to make full use of this tool. I’ve gone through the workflow line by line, it seems to work well. However, authors can improve their work by addressing the following issues.\n\nThere should be an R code script which includes all source code and concise comments like the one in company with the vignette in any Bioconductor package. It would be much easy for the users/reviewers to try the example code. It is not convenient to follow the code in this manuscript, the code need to be edit to remove the prompt symbols (> or +) at each line when copying/pasting.\n\nIt takes too long to run the egsea analysis example on modest machine. It is advisable to show a lesser example in the workflow with only one gene set collection like kegg and just a few base methods like:\ngsa = egsea(voom.results=v, contrasts=contr.matrix,\n\ngs.annots=gs.annots$kegg, symbolsMap=symbolsMap,\n\nbaseGSEAs=baseMethods[1:4], sort.by=\"med.rank\",\n\nnum.threads = 3, report = FALSE)\n\nThe rank of the gsa results shown following the t = topSets(..) line is confusing. The p.adj for the top 1 gene set is not the smallest, actually much bigger than top 2, 6 and 8. Presumably, the gene sets are ranked by med.rank instead of p.adj here. However, the opposite was described in the text above near the egsea.sort() line: “Although p.adj is the default option for sorting EGSEA results for convenience, ...”\n\nIn addition, there is big difference between the final rank and med.rank (e.g. 1 vs 36). This may suggest inconsistent results came from different base methods. This may also be due to the large number of gene sets being tested. Again, using a smaller gene set collection and a few base methods could make the ranking more consistent.\n\nAll visualization functions, i.e. plotHeatmap, plotPathway, plotGOGraph, plotMethods, plotSummary and plotBars share largely the same set of arguments, they can have a unified wrapper function like plot.gsa() with an extra argument type to specify the plot type.\n\nFunctions plotPathway, plotGOGraph are wrapper functions for those in the pathview and topGO package as the author noted in the text. It would be good to explicit show some message like “calling plotting function from pathview or topGO package etc”, just like the message when running egsea().\n\nHTML report of the results is a very valuable feature for the users. However, the code can run a long time, it would be helpful to add some progress reminder message to generateReport() function like egsea(). BTW, the KEGG Pathway graphs are not shown properly in the report example at http://bioinf.wehi.edu.au/EGSEA/mam-rnaseq-egsea-report/index.html.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Partly\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] }, { "id": "27967", "date": "20 Dec 2017", "name": "Pekka Kohonen", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nGSEA analysis methods do not all produce same results. Score-based gene set analysis methods like the Broad Institute GSEA tool are considered to perform better than normal Fisher’s exact test (overrepresentation analysis). But analysts often use methods they know to be less than ideal in order to reduce complexity and save time. So it is good to have a unified interface for GSEA analyses with R – it helps save programming time and reduces complexity. In addition EGSEA is a unique method that combines up to 12 gene set analysis methods into a single score. Independent test also corroborate that the tool using the 12 has more specificity and good sensitivity compared to using some of the tests alone.\nThe EGSEA 1-2-3 workflow is easy to use and generate good-quality figures with the ggplot2 R package. So9me of the figures are novel compared to other packages e.g., scatter plots designed to compare different contrasts. It is also very useful that the tool can be applied to multiple contrasts at a time, although if there are too many contrasts then the number of plots becomes unwieldly (increases combinatorically).\nSome more technical comments: The results object is very complicated for retrieving individual method analysis results (although summaries are readily available). I quite like the \"biobroom\" Bioconductor package that does \"tidy\" data frames from limma results objects.\nAll in all a very useful package both for automating the running of lots of methods at the same time and of course for the \"ensemble\" method. It is recommended to be considered to be part of a standard bioinformatics workflow.\n\nIs the rationale for developing the new software tool clearly explained? Yes\n\nIs the description of the software tool technically sound? Yes\n\nAre sufficient details of the code, methods and analysis (if applicable) provided to allow replication of the software development and its use by others? Yes\n\nIs sufficient information provided to allow interpretation of the expected output datasets and any results generated using the tool? Yes\n\nAre the conclusions about the tool and its performance adequately supported by the findings presented in the article? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-2010
https://f1000research.com/articles/6-1996/v1	13 Nov 17	{ "type": "Research Note", "title": "Anaesthesia for open wrist fracture surgery in adults/elderly", "authors": [ "Irene Sellbrandt", "Metha Brattwall", "Margareta Warrén Stomberg", "Pether Jildenstål", "Jan G. Jakobsson", "Irene Sellbrandt", "Metha Brattwall", "Margareta Warrén Stomberg", "Pether Jildenstål" ], "abstract": "Anaesthetic technique for open surgery of acute distal for arm fracture in adults/elderly is not well defined. Regional anaesthesia, general anaesthesia or a combined general and regional block may be considered. General anaesthetic technique, the timing and drug/drug combination for the regional block must also be considered. This is a study around published studies assessing anaesthtic technique for wrist surgery. A systematic database search was performed and papers describing the effect of anaesthetic techniques were included. We found sparse evidence for what anaesthetic technique is optimal for open wrist fracture repair. In total only six studies were found using our inclusion criteria, which all supported the short term, early recovery benefits of regional anaesthesia as part of multi-modal analgesia. More protracted outcomes and putting the type of block into context of quality of recovery and patients’ satisfaction is lacking in the literature. The risk for a pain rebound when the block vanishes should also be acknowledged. Therefore, further high quality studies are warranted concerning the anaesthetic technique for this type of surgery.", "keywords": [ "open wrist surgery", "anaesthetic technique", "regional anaesthesia", "general anaesthesia", "postoperative pain" ], "content": "Introduction\n\nFracture on the upper limb is common, and fracture on the distal forearm, wrist fracture, is one of most common upper limb fractures. Whether the incident increases or declines at least among women is a matter of discussion1,2. The incidence is increasing among adults and elderly in Sweden3. There are certainly several factors contributing to the increased risk; the fact that individuals are becoming more active at later stages of life and osteoporosis is prevalent in older individuals are both likely to have importance. In addition, there is seasonal variation of distal wrist fracture with high risk during winter months4,5.\n\nThe best treatment of wrist fracture is debated. Data from the Swedish fracture register1,6 shows that approximately 20% of acute wrist fractures undergo open surgery. In 2009, Smektala et al.7 conducted a cohort study around surgical wrist fracture care in Germany. They found that most patients were elderly females, and the predominant fracture management procedure was percutaneous K-wire osteosynthesis (56% of cases), followed by plate osteosynthesis (44%) and more than half of patients had general anaesthesia (55%).\n\nSurgical repair of wrist fracture can be performed under various anaesthetic techniques. Studies assessing anaesthetic technique, whether regional anaesthesia, general anaesthesia or a combined general and regional anaesthetic approach provides the best perioperative care are not well defined. Currently, there is no firm evidence to support a best technique and no clear consensus guidance is available.\n\nThe aim of this study was to identify the currently available data regarding anaesthetic technique, general, regional or combined general and regional anaesthesia, and postoperative pain course for open surgical repair of wrist fracture in adults and elderly.\n\n\nMethods\n\nA systematic database search was performed on PubMed, Scopus and Cochrane databases, searching for articles in the period from 2007 to October 2017 (the last 10 year period), to identify studies regarding the impact of anaesthesia method on pain and long term outcomes after open radius fracture repair. Quantitative as well as qualitative English language studies limited to adults were searched.\n\nThe search strategy used the following text words or Medical Subject Headings (MeSH): “Radius fractures, Wrist injuries, Surgery, Anaesthesia, Regional anaesthesia, Pain, Postoperative, Clinical outcome”, combined search for the last 10 years; (radius fractures OR wrist injuries OR wrist fractures) AND anesthesia. The result was further analysed by two of the authors and secondary papers searched from the initial studies retrieved.\n\nObservational studies and case series were excluded, as it was considered that these could be associated with major bias. Study selection was based on an initial screen of titles and abstracts, and then reading the final identified articles. Papers with at least two different techniques assessing clinical peroperative effects were included.\n\nSelection of papers and subsequent extraction was performed independently by two of the authors (MWS and JGJ). Consensus was reached for final article inclusion and data extraction through discussion between all authors.\n\nA standard data extraction matrix was conducted manually, to gather study information including: references/year of publication/author, anaesthesia method, postoperative pain before and after discharge from hospital.\n\n\nResults\n\nFigure 1 describes the search results from the literature.\n\nIn total, 6 studies, 4 prospective randomised and 2 retrospective studies of various designs, were identified, which altogether included 619 participants (prospective, n=238; retrospective, n=381) (see Table 1).\n\nGA, general anaesthesia; RA, regional anaesthesia; IRA, intravenous regional anaesthesia (Bier block).\n\nOverall, it could be seen that there is little information around anaesthetic techniques used, and base pain medications is sparsely addressed. The focus has been on early and intermediate pain and opioid consumption. Pain was assessed with different scales in each study.\n\nHadzic et al.8 performed a study in 2004 comparing general anaesthesia to infraclavicular plexus block with short-acting local anaesthesia for hand and wrist surgery. They included 52 patients; however 2 were excluded from analysis, resulting in 50 patients; 25 each in general and regional anaesthesia were evaluated. The focus was on early recovery during the stay in hospital, but patients were also called on the telephone once daily for days 1, 2 and 3 and interviewed about pain felt. In addition, 2 weeks later the patients were asked about satisfaction and willingness to have the same anaesthesia. They found that the block group needed approximately 5 minutes longer time for preparation prior to surgery, but more patients in the block group were eligible for early recovery room bypass. The block group also experienced less pain, nausea, sore throat and fatigue. The block group were eligible for discharge at a significantly faster rate that the anaesthesia group. Pain at follow-up did not differ at day 1 and 3 between the two groups, but was found in favour, lower pain, for block group of patient at day 3. Satisfaction with anaesthesia at 2 weeks was found to be the same, but willingness to have same anaesthesia favoured the block technique.\n\nGalos et al.9 randomized patients to general anaesthesia (n=18) or plexus block (n=18) and followed postoperative pain 2, 4, 6, 12, 24, 48 and 72 hours after surgery. They found that the general anaesthesia group had more pain 2 hours postoperatively, but regional anaesthesia experienced more pain 12 and 24 hours after surgery, termed “rebound pain”, as compared to the general anaesthesia patients. There were no differences in pain ratings at 48 and 72 hours or at 2 weeks. Follow-up of function at 2 and 6 weeks did not show any differences.\n\nO’ Neil et al.10 studied 98 patients, 45 receiving general anaesthesia and 53 regional anaesthesia, a single shot peripheral block, for preoperative management. They had a questionnaire follow-up at a 2-week follow-up visit where the amount of pain medication used was requested. They did not find any difference in opioid need between general and regional anaesthesia groups. Opioid need decreased, however, with age and increased in relation to the severity of the fracture.\n\nHolmberg et al.11 studied 52 patients with fractured radius. The study compared pre- and post-operative block, in both groups combined with general anaesthesia total intravenous anaesthesia (TIVA). The patients were randomized to a preoperative (n=25) or a postoperative (n=26) infraclavicular plexus block. Time to the first rescue analgesic, as well as pain score evaluation, was at 1, 2, 4, 8 and 24 hours after surgery. Patients were further called for phone interview on day 7 and 6 months postoperatively, and asked about pain, rescue analgesia daily function and side effects. The time for needing first rescue opioid analgesic was significantly longer for the pre-operative block group (544 vs 343 minutes). There was also a significantly lower mean VAS pain score at 30 minutes, 1, 2 and 4 hours. Pain was, however, not different after 4 hours and beyond. At day 7 more patients in the postoperative block group needed oral paracetamol as compared to the preoperative group; however, the total opioid need was the same. During the first postoperative night, most patients from both groups experienced severe pain. Follow-up at 6 months showed no difference in pain; median pain score in the pre-operative group was 2 as compared to the postoperative group median of 1.\n\nEgol et al.12 published a retrospective register study in 2012 assessing pain after wrist surgery. Included in the analysis were 122 patients that had surgery under general anaesthesia and 65 under a regional block. Patients were followed up for analysis at 3, 6 and 12 months after surgery; pain and function was assessed. At 3 and 6 months, patient that had regional anaesthesia showed significantly less pain and better function; however, at 12 months groups were equal in pain assessment, and function measured by the test were similar and normalised, back to normal range.\n\nSunderland et al.13 conducted a retrospective review, for a retrospective quality improvement project, to assess the need for unplanned medical visits caused by pain in the first 48 hours after wrist surgery and the impact of anaesthetic technique, general anaesthesia or regional block. All 77 patients who had general and 118 patients who had regional anaesthesia were reached for follow-up interview within 6 weeks, which posed the question “In the first 24 to 48 hours after surgery, did you need to seek medical attention for pain?”. The block group was found to have significantly more unplanned visits (20% vs 5%), and self-reported severe pain was also more common in this group of patients (41% vs 10%), as compared to the general anaesthesia group of patients. Patients were also asked about persistent post-operative pain and if they had been diagnosed with a “complex regional pain syndrome” (CRPS). No difference in development of chronic pain or diagnosis of CRPS was found between the two groups.\n\n\nDiscussion\n\nMost wrist fractures in adults and the elderly can be managed by close reposition. However, certain fractures require open reposition and fixation at initial examination, and others fail close reposition and fixation therapy and thus require an open procedure. Wrist surgery is not uncommonly performed as day or over-night stay surgery and is common in elderly patients. Thus, it is of importance to consider various outcomes when comparing anaesthetic techniques used in these procedures and on these patients.\n\nWe found only six papers explicitly addressing anaesthetic technique for open acute distal arm surgery. The pharmacological effect of blocking nociceptive influx is clear; preoperative block seems better than post-surgery block. The effect beyond the early period and duration of the pharmacological blockade was more diverse between studies, and it seems that the risk for “rebound” pain should be acknowledged.\n\nThe benefits of prevention of pain and the concept of multi modal analgesia have been known for decades14. The importance of multi-modal analgesia is well-accepted, and today this standard of care is especially important in ambulatory surgery15. The application of local anaesthetics in the wound area, site for incision, or as a peripheral block, is a basic part of the multi-modal analgesia concept. The effects of peripheral block on early pain, reducing the need for opioid analgesics are well-known16. The effects beyond the early period and the duration of the pharmacological local block is less well documented17. There are several options to improve the duration by the addition of adjuncts e.g. alf-2-blocking agents, such as clonidine or dexmedetomidine; however, the prolongation of the block resolution by liposomal bupivacaine preparation needs further studies18.\n\nHadzic et al. used propofol followed by desflurane and Holberg et al. describes general anaesthesia as TIVA for all patients, the general anaesthetic techniques used were not further addressed. Awakening/emergence from anaesthesia is faster when inhaled anaesthesia has been used, shown by many previous studies19,20. The benefits of the use of nitrous oxide to quicken emergence is also well documented21,22. The impact of low blood gas solubility on awakening is also shown with xenon as the main anaesthetic23. Xenon does promote stable intraoperative haemodynamic and rapid recovery, as compared to traditional halogenated inhaled anaesthetics24. The classic study by Apfel et al. verified the importance of multi-modal PostOperative Nausea and Vomiting - PONV prophylaxis25. Appropriate risk based PONV prophylaxis should be administered reducing its occurrence26. The benefits of propofol-based anaesthesia to reduce early PONV is well known27.\n\nFrom the six studies identified, the blocks were done with different techniques and solutions. The studies by Hadzic et al., Egol et al., Galos et al. and Holmberg et al. used sole infraclavicular block technique, while Sunderland et al. used mixed infra- and supraclavicular block technique and O’Neil et al. used sole supraclavicular blocks. The local anaesthetics used differed as well: Hadzic et al, chloroprocaine; Galos et al, mixed lidocaine and bupivacaine; O’Neil et al., ropivacaine and dexamethasone; Holmberg, ropivacaine. Adding adjuncts to increase the analgesic block duration is of interest, but generally is off-label use, and there is no firm consensus about what drug and concentration should be usedxiii. There is a recent review suggesting dexmedetomidine is superior to clonidine as adjunct for supraclavicular block, but there is also a risk of side effects28.\n\nWe did not address anaesthesia/analgesia/sedation for closed reduction; there is a Cochrane review around anaesthesia for the close reposition from 200229. Pain associated with the performance of local anaesthetic blockade was not evaluated in this study but may be further taken into consideration in the patient´s participation in anaesthetic decision.\n\nIt is obvious that further high quality studies are warranted; studies assessing not only early but more protracted outcome variables. For example, effect on logistics, theatre time, patients eligible for fast-track, bypassing recovery area and time to discharge should all be included. Effects beyond discharge need to also be assessed in a standardised fashion, pain and analgesic requirement at least up to a week post-surgery and quality of recovery should be assessed by a standardised tool. There are today at least two well-accepted tools for the assessment of quality of recovery, the PostopQRS and the Quality of Recovery scale30. The long-term outcome and the risk for chronic pain is dependent on several factors and the impact on anaesthetic technique may not have major impact. Long term outcomes are of importance, but the impact on training, rehabilitation surgical technique and even osteoporosis must be acknowledged when assessing long-term results. Similarly, considerations must be taken that wrist fracture may initiate complex regional pain syndrome type I. For instance, severe fractures and women are at high risk for development this syndrome after surgical treatment of distal radius fractures (Roh et al.., 2014)31.\n\nIn conclusion, there is sparse evidence for what anaesthetic technique is superior for open wrist repair. The short term, early recovery benefits of regional anaesthesia as part of multi-modal analgesia is well documented; however, more protracted outcomes and putting the type of block into context of quality of recovery and patients’ satisfaction is lacking. The risk for pain rebound when the block finishes should also be acknowledged. Further high quality studies are warranted.\n\n\nNotes\n\n1https://stratum.registercentrum.se/#!page?id=1094", "appendix": "Competing interests\n\n\n\nNo competing interests were disclosed.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nReferences\n\nAbrahamsen B, Jørgensen NR, Schwarz P: Epidemiology of forearm fractures in adults in Denmark: national age- and gender-specific incidence rates, ratio of forearm to hip fractures, and extent of surgical fracture repair in inpatients and outpatients. Osteoporos Int. 2015; 26(1): 67–76. PubMed Abstract \| Publisher Full Text\n\nDimai HP, Svedbom A, Fahrleitner-Pammer A, et al.: Epidemiology of distal forearm fractures in Austria between 1989 and 2010. Osteoporos Int. 2014; 25(9): 2297–306. PubMed Abstract \| Publisher Full Text\n\nJerrhag D, Englund M, Karlsson MK, et al.: Epidemiology and time trends of distal forearm fractures in adults - a study of 11.2 million person-years in Sweden. BMC Musculoskelet Disord. 2017; 18(1): 240. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHoff M, Torvik IA, Schei B: Forearm fractures in Central Norway, 1999–2012: incidence, time trends, and seasonal variation. Arch Osteoporos. 2016; 11: 7. PubMed Abstract \| Publisher Full Text\n\nOmsland TK, Ahmed LA, Grønskag A, et al.: More forearm fractures among urban than rural women: the NOREPOS study based on the Tromsø study and the HUNT study. J Bone Miner Res. 2011; 26(4): 850–6. PubMed Abstract \| Publisher Full Text\n\nHorst TA, Jupiter JB: Stabilisation of distal radius fractures: Lessons learned and future directions. Injury. 2016; 47(2): 313–9. PubMed Abstract \| Publisher Full Text\n\nSmektala R, Endres HG, Dasch B, et al.: [Quality of care after distal radius fracture in Germany. Results of a fracture register of 1,201 elderly patients]. Unfallchirurg. 2009; 112(1): 46–54. PubMed Abstract \| Publisher Full Text\n\nHadzic A, Arliss J, Kerimoglu B, et al.: A comparison of infraclavicular nerve block versus general anesthesia for hand and wrist day-case surgeries. Anesthesiology. 2004; 101(1): 127–32. PubMed Abstract \| Publisher Full Text\n\nGalos DK, Taormina DP, Crespo A, et al.: Does Brachial Plexus Blockade Result in Improved Pain Scores After Distal Radius Fracture Fixation? A Randomized Trial. Clin Orthop Relat Res. 2016; 474(5): 1247–54. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nO'Neil JT, Wang ML, Kim N, et al.: Prospective Evaluation of Opioid Consumption After Distal Radius Fracture Repair Surgery. Am J Orthop (Belle Mead NJ). 2017; 46(1): E35–E40. PubMed Abstract\n\nHolmberg A, Sauter AR, Klaastad Ø, et al.: Pre-operative brachial plexus block compared with an identical block performed at the end of surgery: a prospective, double-blind, randomised clinical trial. Anaesthesia. 2017; 72(8): 967–977. PubMed Abstract \| Publisher Full Text\n\nEgol KA, Soojian MG, Walsh M, et al.: Regional anesthesia improves outcome after distal radius fracture fixation over general anesthesia. J Orthop Trauma. 2012; 26(9): 545–9. PubMed Abstract \| Publisher Full Text\n\nSunderland S, Yarnold CH, Head SJ, et al.: Regional Versus General Anesthesia and the Incidence of Unplanned Health Care Resource Utilization for Postoperative Pain After Wrist Fracture Surgery: Results From a Retrospective Quality Improvement Project. Reg Anesth Pain Med. 2016; 41(1): 22–7. PubMed Abstract \| Publisher Full Text\n\nMichaloliakou C, Chung F, Sharma S: Preoperative multimodal analgesia facilitates recovery after ambulatory laparoscopic cholecystectomy. Anesth Analg. 1996; 82(1): 44–51. PubMed Abstract \| Publisher Full Text\n\nWarren-Stomberg M, Brattwall M, Jakobsson JG: Non-opioid analgesics for pain management following ambulatory surgery: a review. Minerva Anestesiol. 2013; 79(9): 1077–87. PubMed Abstract\n\nJakobsson J, Johnson MZ: Perioperative regional anaesthesia and postoperative longer-term outcomes [version 1; referees: 3 approved]. F1000Res. 2016; 5: pii: F1000 Faculty Rev-2501. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nBrattwall M, Jildenstål P, Warré Stomberg M, et al.: Upper extremity nerve block: how can benefit, duration, and safety be improved? An update [version 1; referees: 3 approved]. F1000Res. 2016; 5: pii: F1000 Faculty Rev-907. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nHamilton TW, Athanassoglou V, Mellon S, et al.: Liposomal bupivacaine infiltration at the surgical site for the management of postoperative pain. Cochrane Database Syst Rev. 2017; 2: CD011419. PubMed Abstract \| Publisher Full Text\n\nDexter F, Tinker JH: Comparisons between desflurane and isoflurane or propofol on time to following commands and time to discharge. A metaanalysis. Anesthesiology. 1995; 83(1): 77–82. PubMed Abstract \| Publisher Full Text\n\nWachtel RE, Dexter F, Epstein RH, et al.: Meta-analysis of desflurane and propofol average times and variability in times to extubation and following commands. Can J Anaesth. 2011; 58(8): 714–24. PubMed Abstract \| Publisher Full Text\n\nJakobsson I, Heidvall M, Davidson S: The sevoflurane-sparing effect of nitrous oxide: a clinical study. Acta Anaesthesiol Scand. 1999; 43(4): 411–4. PubMed Abstract \| Publisher Full Text\n\nEinarsson S, Bengtsson A, Stenqvist O, et al.: Decreased respiratory depression during emergence from anesthesia with sevoflurane/N2O than with sevoflurane alone. Can J Anaesth. 1999; 46(4): 335–41. PubMed Abstract \| Publisher Full Text\n\nRasmussen LS, Schmehl W, Jakobsson J: Comparison of xenon with propofol for supplementary general anaesthesia for knee replacement: a randomized study. Br J Anaesth. 2006; 97(2): 154–9. PubMed Abstract \| Publisher Full Text\n\nLaw LS, Lo EA, Gan TJ: Xenon Anesthesia: A Systematic Review and Meta-Analysis of Randomized Controlled Trials. Anesth Analg. 2016; 122(3): 678–97. PubMed Abstract \| Publisher Full Text\n\nApfel CC, Korttila K, Abdalla M, et al.: A factorial trial of six interventions for the prevention of postoperative nausea and vomiting. N Engl J Med. 2004; 350(24): 2441–51. PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nÖbrink E, Jildenstål P, Oddby E, et al.: Post-operative nausea and vomiting: update on predicting the probability and ways to minimize its occurrence, with focus on ambulatory surgery. Int J Surg. 2015; 15: 100–6. PubMed Abstract \| Publisher Full Text\n\nKumar G, Stendall C, Mistry R, et al.: A comparison of total intravenous anaesthesia using propofol with sevoflurane or desflurane in ambulatory surgery: systematic review and meta-analysis. Anaesthesia. 2014; 69(10): 1138–50. PubMed Abstract \| Publisher Full Text\n\nEl-Boghdadly K, Brull R, Sehmbi H, et al.: Perineural Dexmedetomidine Is More Effective Than Clonidine When Added to Local Anesthetic for Supraclavicular Brachial Plexus Block: A Systematic Review and Meta-analysis. Anesth Analg. 2017; 124(6): 2008–2020. PubMed Abstract \| Publisher Full Text\n\nHandoll HH, Madhok R, Dodds C: Anaesthesia for treating distal radial fracture in adults. Cochrane Database Syst Rev. 2002; (3): CD003320. PubMed Abstract \| Publisher Full Text\n\nBowyer A, Jakobsson J, Ljungqvist O, et al.: A review of the scope and measurement of postoperative quality of recovery. Anaesthesia. 2014; 69(11): 1266–78. PubMed Abstract \| Publisher Full Text\n\nRoh YH, Lee BK, Noh JH, et al.: Factors associated with complex regional pain syndrome type I in patients with surgically treated distal radius fracture. Arch Orthop Trauma Surg. 2014; 134(12): 1775–81. PubMed Abstract \| Publisher Full Text" }	[ { "id": "28244", "date": "11 Dec 2017", "name": "Jan Hendrik Eshuis", "expertise": [ "Reviewer Expertise Anesthesiologist", "expert in regional anesthesia as well as in ambulatory surgery and anesthesia" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe article’s title suggests focusing on the elderly but the studies mentioned do not refer especially to the elderly. Although the authors are right in saying that wrist fractures are more common in the elderly it is not clear in the studies mentioned; I suggest to remove “elderly” from the title. In the text it is rightly mentioned.\nIt is interesting that the article shows a relative lack of information in the literature on this specific wrist fracture treatment. I agree with all the conclusions drawn by the authors about desired further studies; important that is noted that rebound pain in the aftercare phase is higher in the regional group than the multimodal group of patients; this provides us an important “mirror” to our daily practice!\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] }, { "id": "29364", "date": "16 Jan 2018", "name": "Colin F. Royse", "expertise": [], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe authors have conducted a systematic review to identify if regional versus general anaesthesia is superior for post-operative pain for distal wrist fractures. They correctly show that there is an evidence gap in the literature, and cannot provide recommendation from their data. The study is well conducted, but the contributing trials are all small and have sufficient heterogeneity that it is simply not possible to draw any conclusions, other than more trials need to be done.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate? Not applicable\n\nAre all the source data underlying the results available to ensure full reproducibility? No source data required\n\nAre the conclusions drawn adequately supported by the results? Yes", "responses": [] } ]	1	https://f1000research.com/articles/6-1996
https://f1000research.com/articles/6-1995/v1	13 Nov 17	{ "type": "Research Article", "title": "Analyzing DECREASE trials to estimate evidence of data manipulation", "authors": [ "Chris H.J. Hartgerink", "E.M. Kemper", "Markus W. Hollmann", "Gerben ter Riet", "E.M. Kemper", "Markus W. Hollmann", "Gerben ter Riet" ], "abstract": "Background: The effect of beta-blockers on perioperative mortality in non-cardiac surgery has been controversial due to concerns regarding the scientific integrity of the DECREASE-I and DECREASE-IV trials. Previous meta-analyses indicated beta-blockade might increase mortality after removing the DECREASE trials from the evidence base. Methods: In this report, we statistically investigate the DECREASE trials and model their veracity (i.e., the probability that these effects or more extreme occurred naturally) and estimate how many data points might have been manipulated in the DECREASE trials using the inversion method. Results: Our research indicates that the DECREASE trials are nearly impossible if we assume they investigate the same effect as the non-DECREASE trials and under that assumption. Our results also provide evidence that at least some data points were manipulated. Conclusions: The DECREASE trials are likely to be manipulated under the assumption that they investigate the same effect as the non-DECREASE trials on beta-blockade. However, these differences might also be due to different conceptual approaches as to how beta-blockade might prevent mortality in non-cardiac surgery. Considering this, we recommend new and more extensively controlled, confirmatory trials to determine whether there is any use in administering beta-blockers in order to decrease perioperative mortality.", "keywords": [ "beta-blocker", "DECREASE", "data manipulation", "inversion method", "misconduct" ], "content": "Introduction\n\nThe effect of beta-blockers on perioperative mortality in non-cardiac surgery has been controversial1 due to concerns regarding the scientific integrity in two related clinical trials2–6. Three meta-analyses that included the trials subject to concerns concluded that beta-blockers decrease perioperative mortality7–9, whereas a meta-analysis that excluded the suspect trials concluded that beta-blockers increase perioperative mortality9. In these studies, perioperative mortality was defined as the death rate of patients in the perioperative setting, including the period of admission, anaesthesia with surgery, and postoperative recovery.\n\nThe trials subject to concerns regarding scientific integrity were the Dutch DECREASE-I and DECREASE-IV trials4–6. The committees that investigated the integrity of the DECREASE trials reported that data manipulation was likely, but that the extent of the data manipulation remained unclear4–6. Moreover, the latest guidelines still recommend the usage of beta-blockers in the perioperative period in certain cases10,11, where some of these guidelines are based on other work by the PI of the DECREASE trials. Considering the potential harmful consequences of guidelines based on work by someone who has been repeatedly investigated for breaching scientific integrity, we aim to estimate the extent of data manipulation in the DECREASE studies2–6 to further stimulate the debate on using beta-blockers in the perioperative period for patients undergoing non-cardiac surgery.\n\nThe reports on the integrity of the DECREASE trials primarily focused on the provenance of the raw data but did not investigate the extent to which the DECREASE trials deviated from comparable trials. Provenance is primarily concerned with the origins of the data, verifying things such as (but not limited to) the informed consent and whether data correspond to patient files. However, the committee reports did not neglect statistical evaluation; according to the report a statistical expert assessed the applicability of forensic statistical methods6 to evaluate results of trials separately (i.e., DECREASE-I, DECREASE-IV), although the report lacks details as to how this evaluation took place. The expert concluded that the methods previously applied by him were not applicable for use in this case. Nonetheless, in the present study we compare across trials, which is a method that has previously been used to monitor trial data quality or to test for potential data anomalies12,13. Moreover, comparing across trials instead of evaluating them separately has previously proven to be effective in detecting data manipulation14. Comparing the DECREASE trials to other published trials studying the effectiveness of beta-blockers with respect to perioperative mortality could prove informative of the potential extent of the manipulation in the DECREASE trials.\n\nThe effectiveness of perioperative beta-blockade is obfuscated by the afflicted DECREASE trials, potentially interacting with the type of beta-blocker and the way that beta-blockers were administered (i.e., dose and duration of treatment). In the randomized trials on beta-blockers, patients were administered various types of beta-blockers (e.g., metroprolol, bisoprolol, atenolol) and in various ways (e.g., intravenously, orally; half an hour before surgery or multiple days before surgery; with or without titration based on heart rate). Factors such as dosage and duration can have an effect on the pharmacological effectiveness with respect to perioperative mortality. Moreover, the highly discrepant results from the DECREASE trials9 might partly be caused by such differences15 and not purely due to data manipulation (given that not all data points can be considered manipulated at this point).\n\nTo statistically investigate the evidence for data manipulation in the DECREASE studies2,3, we took three steps. First, we reproduced the findings from the 2014 meta-analysis by Bouri et al.9, which contained sufficient information to estimate the deviation of the DECREASE trials from other published trials on beta-blockers. We also included type of beta-blocker to inspect whether this is predictive of the effect of beta-blockers on perioperative mortality. Second, we evaluated the probability that the DECREASE trials (or more extreme effects) arose from the same effect distribution as the non-DECREASE trials, which are assumed to be the true effect of beta-blockers on perioperative mortality in patients undergoing non-cardiac surgery. Third, we estimated how many data points would have to be manipulated in order to reproduce the results of the DECREASE trials if the initial, non-manipulated results arose from the effect estimates as obtained from the non-DECREASE trials. Considering the committees investigating the scientific integrity of the DECREASE trials were unable to assess this, we consider it worthwhile to investigate this further.\n\n\nStep 1: Reproducing meta-analysis of Bouri et al. (2014)\n\nTo ensure that we used similar analysis procedures as in the 2014 meta-analysis9, we initially reproduced Bouri et al.'s estimates. This ensured that (1) their results are reproducible and (2) we are using the correct estimates in subsequent steps of our analyses. Using Figure 2 and Figure 3 from the original paper9, we extracted the raw event data for the 2 (control vs experimental) by 2 (event vs no event) design, which we used to recompute the natural logarithm of the risk ratio and its standard error. The extracted event data is available at osf.io/aykeh and our analysis plan was preregistered at osf.io/vnmzc.\n\nWe computed the log risk ratio (i.e., log RR) for each study and pooled these using v2.0.0 of the R package metafor16. We estimated a weighted random-effects model using the restricted maximum-likelihood estimator (i.e., REML)17 to estimate the variance of effects. We used the default weighting procedure in the metafor package. We added 0.5 to each cell count, as is common in meta-analyses on risk- and odds ratios in order to prevent computational artefacts18. The 2014 meta-analysis9 did not specify the variance estimate used; hence, (minor) discrepancies between our estimates and the original estimates could be due to differences in the estimation procedure.\n\nWe were able to closely reproduce the estimates for the different sets of studies (Figure 2 of the 2014 meta-analysis9). Bouri et al. differentiated between the estimates from the non-DECREASE trials (k = 9) and the DECREASE trials (k = 2). We confirmed the effect size estimates and the variance estimates for both the non-DECREASE and the DECREASE trials, except for some discrepancies at the second decimal level for the estimated effect sizes and a somewhat larger difference between the variance estimates of the DECREASE studies. Table 1 depicts the original and reproduced values for both sets of studies.\n\nSecond, we meta-analyzed all studies combined, including a dummy predictor for the DECREASE and non-DECREASE studies to reproduce results presented in Figure 4 of the 2014 meta-analysis9. Our results showed a bit more evidence against equal subgroups than the original meta-analysis9 (original: χ2(1) = 3.91, p = .05; reproduced: χ2(1) = 6.12, p = 0.013). Additionally, the original analyses showed substantial residual heterogeneity (I2 = 74.4%), whereas we found no residual heterogeneity (I2 = 0%). Different variance estimates (e.g., DerSimonian-Laird instead of REML) did not resolve this difference. We tried to clarify these discrepancies by e-mailing the original authors (including a reminder after several weeks), but did not receive a response. Nonetheless, the broad strokes of the meta-regression confirmed that the DECREASE trials were the determining predictor for the effectiveness of beta-blockers (including DECREASE: RR = 0.509; excluding DECREASE: RR = 1.275).\n\nAdditionally, and exploratively, we evaluated the predictive effect of the type of beta-blocker used in the trials. Descriptively, the DECREASE trials remained predictive of decreased mortality (RR = 0.509), whereas the non-DECREASE trials provide tentative evidence that atenolol results in lower mortality (RR = 0.777). Nonetheless, for other beta-blockers in the non-DECREASE trials, there is descriptive evidence that beta-blockers could increase mortality (bisoprolol: RR = 2.973; metoprolol: RR = 1.303; propranolol: RR = 1.7). Table 2 shows the meta-regression results in full. We do note that the DECREASE studies only use bisoprolol and any estimates for other beta-blockers are extrapolations.\n\n\nStep 2: Evaluating the veracity of DECREASE studies\n\nBased on the effect estimates for the non-DECREASE trials from Step 1, we estimated the probability that the observed effects from the DECREASE studies (or more extreme) occurred naturally. We assumed that the non-DECREASE studies estimated the true effect distribution of perioperative beta-blockade on mortality, not perturbed by publication bias due to statistical (non)significance. Publication bias was assumed to not be a problem because a substantial number of nonsignificant effects are included in the dataset (9 of 11 results are nonsignificant). Based on this effect distribution, we estimated the veracity of the DECREASE trials separately, which is the estimated probability of the observed data (or more extreme) under a given true effect19.\n\nBased on the estimated effect distribution from the non-DECREASE trials, we calculated the probability of each DECREASE trial result, or a more extreme result. In other words, we computed the two-tailed p-value for the null hypothesis that the DECREASE trials arose from the same effect distribution as the non-DECREASE trials (H0: μx¯1−x¯2= 0). To this end, we applied a Welch t-test20. As means, we used the observed log RR for the DECREASE trials (i.e., DECREASE-I: -1.44; DECREASE-IV: -0.452) and the meta-analyzed log RR for the non-DECREASE trials (i.e., 0.243). As standard deviations, we used the standard error for the DECREASE trials (i.e., DECREASE-I: 0.061; DECREASE-IV: 0.018) and the standard error of the estimated log RR for the non-DECREASE trials (i.e., 0.002). We initially preregistered that the DECREASE trials would be regarded as fixed in the computation of the veracity, which was erroneous because these also have their own standard error; hence, we applied the Welch test to take into account the uncertainty in the estimates of both the DECREASE and non-DECREASE trials.\n\nResults indicate that the DECREASE trials are highly unlikely under the estimated effect distribution from the non-DECREASE trials. More specifically, the results from DECREASE-I (or more extreme) have a probability of approximately 1 in 10 000 (t(8) = –6.75, p = 0.000145) and the results from DECREASE-IV (or more extreme) have a probability of approximately 1 in 1000 (t(8) = –4.996, p = 0.0010587). This indicates that the DECREASE trial results are unlikely to have come from the same population effect distribution as the non-DECREASE trials. Moreover, observing two of such extremely unlikely results jointly, as in the DECREASE trials, would occur in only 2 out of 10 million sets of two trials (i.e., 0.0000002) according to this model. Hence, this result indicates that the DECREASE trials are severely different from the non-DECREASE trials.\n\nResults from Step 1 indicated that no between-trial variance (i.e., homogeneity; τ2 = 0) of the effects was observed; given the small number of trials included (i.e., 9), however, this estimate is highly uncertain. The total N across the non-DECREASE trials was 10529. We conducted sensitivity analyses to see how dependent results are on the heterogeneity estimate (not preregistered; osf.io/vnmzc). Fixing the variance estimate τ2 to .5, indicates that the probability of observing the DECREASE trials jointly is approximately 1.2 out of 100 000 (see Figure 1). To put these numbers into context, a variance of 0.25 would suggest that results of perioperative beta-blockade vary substantially due to contextual circumstances of the study, even if perioperative beta-blockade has no effect whatsoever (RRs between 0.779 and 1.284 in ~64% of the cases).\n\n\nStep 3: Estimating the amount of manipulated data\n\nWe estimated the number of data points that would need to be manipulated to arrive at the estimates from the DECREASE trials, given that the non-DECREASE trials represent the true effect of perioperative beta-blockade. In contrast to Step 2, which assumes no data manipulation occurred and that the DECREASE trials occurred naturally from the same effect distribution as the non-DECREASE trials, Step 3 assumes that the DECREASE trials might in fact contain manipulated data. The estimates from Step 3 provide an indication of the extent of potential data manipulation in the DECREASE studies4–6,9.\n\nIn order to estimate the number of manipulated data points, we first estimated the probability of perioperative mortality (in log odds) in each trial arm for each trail stratum. As such, we estimate mortality odds four times: once per condition (beta-blocker or control) per trial stratum (DECREASE- and non-DECREASE trials). For all four combinations of condition and trial type, we ran a meta-analysis applying similar methods used in Step 1, resulting in four meta-analytic absolute mortality estimates with corresponding effect variances. Throughout the simulations, we used the point estimates (i.e., fixed effect) to simulate genuine and manipulated data, but supplemented this by using distribution estimates (i.e., random effects) as sensitivity analyses.\n\nWe applied the inversion method to estimate the number of manipulated data points in the DECREASE trials21. We assumed that if data are manipulated, each data point is manipulated in the same way and to the same extent. The inversion method iteratively hypothesizes that X out of N data points were manipulated (i.e., X = 0,1,..., N), assuming they were manipulated in the same way. For each combination of X and trial, we simulated 10000 datasets. Each simulated dataset contained X manipulated data points and N-X genuine data points. For each simulated dataset (exact simulation procedure in the next paragraph), we determined the likelihood of the results with\n\n\n\nwhere πE indicates the mortality rate in the beta-blocker condition as drawn from the meta-analytic effect distribution (πC indicates the mortality rate in the control condition). We estimated those parameters using the meta-analytic procedure described in the previous paragraph, resulting in the estimates depicted in Table 3. The likelihood was computed under both the manipulated effect estimates (i.e., Lmanipulated) and the genuine data (i.e., Lgenuine). Table 4 indicates which cell sizes the various nXX refer to within the (simulated) data. After computing the likelihoods, we compared them to determine whether the simulated data were more likely to arise from the genuine trials (Lgenuine > Lmanipulated) or from the manipulated trials (Lmanipulated > Lgenuine). Note that comparing the likelihoods is a minor deviation from the preregistration, where we initially planned on using p-value comparisons (osf.io/vnmzc).\n\nWe used these parameters to estimate the number of manipulated data points with the inversion method.\n\nFor each hypothesis of X out of N manipulated data points, we computed the probability that the manipulated data are more likely than the genuine data (pM = P(Lmanipulated > Lgenuine)). Based on pM, we computed the confidence interval for the estimated X manipulated data points (i.e., XLB; XUB). For a 95% confidence interval, the lower bound is equal to the pM closest to .025, whereas the upperbound is equal to the pM closest to .975.\n\nWe computed pM for all X out of N manipulated data points in 10000 randomly generated datasets, which were generated in three steps. For each dataset we:\n\n1. Sampled (across conditions, without replacement) X fictitious participants that would be the result of data manipulation.\n\n2. Determined the population mortality rate for each condition (i.e., for each cell based on the estimates from Table 3). The meta-analytic point estimate was used or a population effect was randomly drawn from the meta-analytic effect distribution.\n\n3. Simulated the number of deaths for the different conditions using a binomial distribution based on the mortality rate as determined in 2, resulting in the cell counts as in Table 4.\n\nBased on the meta-analytic effect from 2 and the cell sizes from 3, we computed the likelihoods Lmanipulated and Lgenuine using Equation 1. As mentioned before, we computed pM, which indicates the probability that the data are more likely under the estimates resulting from the (allegedly) manipulated data (i.e., the DECREASE trials) than under the estimates resulting from the genuine data (i.e., the non-DECREASE trials; pM = P(Lmanipulated > Lgenuine)).\n\nFor DECREASE-I (N = 112), the 95% confidence interval for the estimated number of manipulated data points is [0 – 112] or [0 – 112] when based on a point estimate or a more uncertain distribution estimate, respectively. The left column of Figure 2 depicts the pM per X manipulated data points (top panel) and the bounds of the confidence interval when the degree of confidence is altered (lower panel). Staying clearly between the dotted lines in the top panel, depicting the 95% CI (top: .975; bottom: .025), it becomes apparent that the degree of uncertainty is too high to make any reasonable estimates about the number of manipulated data points with sufficient confidence. This is partly due to the small sample size of the DECREASE-I trial (i.e., N = 112) and the availability of just the summary results. Only when the degree of confidence is lowered to around 75% does the interval not span the entire sample size. As such, based on the summary results, little can be said about the extent of the data manipulation that occurred in the DECREASE-I trial, affirming the conclusions of the original committee report6.\n\nThe top row panels indicate pM (y-axis) for all X out of N manipulated data points (x-axis). The bottom row indicates the estimated interval of manipulated data points (y-axis) when varying the degree of confidence (x-axis). Dotted lines indicate the bounds for a 95 percent CI. The online version of this figure is interactive.\n\nFor DECREASE-IV (N = 1066), the 95% confidence interval for the estimated number of manipulated data points is [3 – 1066] or [10 – 1066] when based on a point estimate or a more uncertain distribution estimate, respectively. The relatively minor difference between the estimates indicates that there is a high degree of confidence that data manipulation did occur based on the difference of the trial results alone. Nonetheless, the range of potentially manipulated data points is still estimated at approximately 1000; this indicates that the summary results are insufficient to provide more than an estimated lower bound. This indicates that it is possible not all data were manipulated (i.e., N = 1066), but at least some were, increasing the importance of well-documented data provenance to discern between genuine and falsified data.\n\n\nDiscussion\n\nThe effect of beta-blockade on perioperative mortality was already unclear based on the investigations regarding scientific integrity; our results strongly affirm that the empirical evidence from the DECREASE trials is highly discrepant from other trials supposedly studying the same effect (i.e., the effectiveness of beta-blockers in decreasing perioperative mortality). Our results indicate that the results from the DECREASE trials are nearly impossible to have arisen from the same effect inspected by the non-DECREASE trials, except when we assume at least some of the data were manipulated. As such, the scientific validity of the DECREASE-I and DECREASE-IV trials should be regarded as highly problematic and untrustworthy when assessing the effectiveness of beta-blockade on perioperative mortality if they truly investigate the same effects as the non-DECREASE trials, as is often assumed9. Nonetheless, the original papers that presented these trial results are not yet retracted2,3, despite the integrity reports4–6.\n\nOur approach to estimating the number of manipulated data points has one major limitation that we would like to highlight: multiplicity. For each estimated proportion of manipulated data points, there is another smaller (or larger) proportion with more (or less) extremely manipulated data points. This problem is similar to how various samples can give rise to the same mean, but contain vastly different individual scores within them (e.g., -2.5 and +2.5 versus -100 and +100; both give the mean zero). Nonetheless, this limitation does still allow us to estimate whether any data manipulation occurred because there is no multiplicity in not manipulating data.\n\nThe ESC/ESA and ACC/AHA guidelines11,22 on perioperative beta-blockade already excluded the DECREASE trials in their assessment, but also explicitly state that other trials by Poldermans are excluded. However, upon close inspection of the reference lists, the ACC/AHA guidelines still cites four trials including Poldermans as author as evidence for the guidelines2,23–25, of which two were already inspected by the scientific integrity committees of Erasmus MC2,23. In the ACC/AHA guidelines, the following is said about studies conducted by Poldermans:\n\n\"If nonretracted DECREASE publications and/or other derivative studies by Poldermans are relevant to the topic, they can only be cited in the text with a comment about the finding compared with the current recommendation but should not form the basis of that recommendation or be used as a reference for the recommendation.\"11\n\nNonetheless, references are made without clear comments. Given the confirmation of problems in the DECREASE-I and DECREASE-IV trials in our results, it stresses that there is reason to distrust trials by Poldermans. For the integrity of the guidelines and the safety of the patients, we pose that investigations should be initiated into works where Poldermans was involved and which were not cleared by the scientific committees of Erasmus MC in their misconduct investigations. In particular those papers cited as evidence in the ACC/AHA guidelines should be investigated, considering that they affect patients and their treatment directly.\n\nPreviously, further investigation of trials by Poldermans was deemed unfeasible due to the lack of raw data; here we indicate methods that do make it feasible. Based on just event-count data and trials that supposedly investigate the same effect, we were able to estimate whether part of the data were in fact manipulated and whether the results were within reason of trials investigating the same effect. The results clearly indicated they were not within such reason.\n\nThe results of our analyses also highlight that, despite the lack of raw data availability, summary results from larger samples allow for more precise estimates of the number of manipulated data points when similar trials are available. Moreover, larger trials result in relatively more certainty (e.g., DECREASE-IV) about the estimated number of manipulated data points, when using the inversion method, compared to smaller trials (e.g., DECREASE-I). This increased certainty is due to decreased standard errors of the estimated effects, resulting in higher sensitivity to data anomalies. Nonetheless, much residual uncertainty remains and simply less information is available in summary results when compared to raw data. As such, raw data availability would improve the options open to detect potential anomalies (note: raw data are available for DECREASE VI, but upon a freedom of information request by the first author, Erasmus MC refused to share these data; see osf.io/zv953/ for original Dutch correspondence). The results also highlight that in order to prevent detection, it would be in the manipulator's interest to fabricate small and imprecise studies (assuming the manipulator wants to remain undetected), which ultimately detracts from the scientific value of such a study and hence the individual reward for manipulation through reduced impact (hopefully).\n\nWith respect to clinical practice, the results provide some tentative evidence that type of beta-blockade can severely influence perioperative mortality. Our reanalysis of the Bouri et al.9 data indicates that type of beta-blockade can reverse the effect on perioperative mortality, even after taking into account whether a study belongs to the DECREASE family. As such, atenolol seems to tentatively decrease perioperative mortality, whereas the others (metoprolol, propranolol, bisoprolol) increase perioperative mortality. However, there seems to be covariation with respect to treatment administration, duration, and dose, which further confounds whether the treatment effect is due to type of beta-blocker or due to one of these other parameters. There are too few studies (k = 11) to properly discern the various treatments from each other, requiring a new randomized trial with high statistical power to determine moderating factors (if any). This affirms the statement from the ESC/ESA guidelines that \"high priority needs to be given to new randomized clinical trials to better identify which patients derive benefit from beta-blocker therapy in the perioperative setting, and to determine the optimal method of beta-blockade\"22.\n\nMoreover, the DECREASE and non-DECREASE trials seem to apply beta-blockade from different conceptual viewpoints that could confound the effectiveness of beta-blockade. The non-DECREASE trials seem to focus purely on the application of beta-blockers in itself, whereas the DECREASE trials use beta-blockade as a proxy to decrease resting heart rate2,3. As such, the DECREASE studies applied beta-blockade at least a week in advance, specifically in order to lower patient's resting heart rate to <70BPM and potentially habituate the patient to the effects of the beta-blockade. Other studies apply the beta-blockade just prior to the surgery (maximum: one day prior), and therefore seem to regard the treatment specifically and not the proxy of lowered BPM. As such, the differences between the DECREASE and non-DECREASE trials might also in part be a consequence of the different approaches in the various trials. Whether these differences matter in treatment decisions is worthy of further research in a clinical trial with high statistical power to find such differences.\n\nIn summary, our research indicates that the DECREASE trials are nearly impossible if we assume they investigate exactly the same effect as the non-DECREASE trials and, under that assumption, our results provide some evidence that at least some data points were manipulated. However, these differences might also be due to different conceptual approaches as to how beta-blockade might prevent mortality in non-cardiac surgery. We recommend renewed investigations into Poldermans’ work given these findings — especially those works still referenced by guidelines on the use of beta-blockers without proper notice. Moreover, it remains unclear whether beta-blockers might be effective in preventing mortality rates in non-cardiac surgery patients. Considering this, we recommend new and more extensively controlled, confirmatory trials to determine whether there is any use in administering beta-blockers in order to decrease perioperative mortality — at the moment there is insufficient evidence to determine any positive effect of beta-blockers on mortality rates.\n\n\nData availability\n\nAll manuscript materials are available at https://github.com/chartgerink/2015poldermans and are preserved at Zenodo (doi.org/10.5281/zenodo.84535426).", "appendix": "Competing interests\n\n\n\nThe authors declare no competing interests.\n\n\nGrant information\n\nCHJH was funded by the Office of Research Integrity during part of this project (HHS-ORI; ORIIR160019).\n\nThe funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\n\nSupplementary material\n\nSupplementary File 1: Session info.\n\nClick here to access the data.\n\n\nReferences\n\nCole GD, Francis DP: Perioperative β blockade: guidelines do not reflect the problems with the evidence from the DECREASE trials. 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PubMed Abstract \| Publisher Full Text \| Free Full Text\n\nESC/ESA: Guidelines on non-cardiac surgery: Cardiovascular assessment and management. Eur Heart J. 2014; 35: 2383–2243. Reference Source\n\nFleisher LA, Fleischmann KE, Auerbach AD, et al.: 2014 ACC/AHA guideline on perioperative cardiovascular evaluation and management of patients undergoing noncardiac surgery: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines. Circulation. 2014; 130(24): 2215–2245. PubMed Abstract \| Publisher Full Text\n\nBuyse M, George SL, Evans S, et al.: The role of biostatistics in the prevention, detection and treatment of fraud in clinical trials. Stat Med. 1999; 18(24): 3435–3451. PubMed Abstract \| Publisher Full Text\n\nCarlisle JB: Data fabrication and other reasons for non-random sampling in 5087 randomised, controlled trials in anaesthetic and general medical journals. Anaesthesia. 2017; 72(8): 944–952. PubMed Abstract \| Publisher Full Text\n\nKnepper D, Lindblad AS, Sharma G, et al.: Statistical monitoring in clinical trials: Best practices for detecting data anomalies suggestive of fabrication or misconduct. Ther Innov Regul Sci. 2016; 50(2): 144–154. Publisher Full Text\n\nvan Klei WA: [Which is the preferred perioperative beta-blocker?]. Ned Tijdschr Geneeskd. 2015; 159: A9798. PubMed Abstract\n\nViechtbauer W: Conducting meta-analyses in R with the metafor package. J Stat Softw. 2010; 36(3): 1–48. Publisher Full Text\n\nViechtbauer W: Bias and efficiency of meta-analytic variance estimators in the Random-Effects model. J Educ Behav Stat. 2005; 30(3): 261–293. Publisher Full Text\n\nAgresti A: Categorical Data Analysis. Hoboken NJ: John Wiley & Sons Inc.; 2002. Reference Source\n\nPeters CFW, Klaassen CAJ, van de Wiel MA: Evaluating the Scientific Veracity of Publications by Dr. Jens Forster. University of Amsterdam; 2015. Reference Source\n\nWelch BL: The generalisation of student’s problems when several different population variances are involved. Biometrika. 1947; 34(1–2): 28–35. PubMed Abstract \| Publisher Full Text\n\nCasella G, Berger RL: Statistical Interference. Pacific Grove, CA:Duxbury; 2002. Reference Source\n\nKristensen SD, Knuuti J, Saraste A, et al.: 2014 ESC/ESA Guidelines on non-cardiac surgery: cardiovascular assessment and management: The Joint Task Force on non-cardiac surgery: cardiovascular assessment and management of the European Society of Cardiology (ESC) and the European Society of Anaesthesiology (ESA). Eur J Anaesthesiol. 2014; 31(10): 517–573. PubMed Abstract \| Publisher Full Text\n\nBoersma E, Poldermans D, Bax JJ, et al.: Predictors of cardiac events after major vascular surgery: Role of clinical characteristics, dobutamine echocardiography, and beta-blocker therapy. JAMA. 2001; 285(14): 1865–73. PubMed Abstract \| Publisher Full Text\n\nvan Kuijk JP, Flu WJ, Schouten O, et al.: Timing of noncardiac surgery after coronary artery stenting with bare metal or drug-eluting stents. Am J Cardiol. 2009; 104(9): 1229–1234. PubMed Abstract \| Publisher Full Text\n\nFlu WJ, van Kuijk JP, Chonchol M, et al.: Timing of pre-operative Beta-blocker treatment in vascular surgery patients: influence on post-operative outcome. J Am Coll Cardiol. 2010; 56(23): 1922–1929. PubMed Abstract \| Publisher Full Text\n\nHartgerink C: chartgerink/2015poldermans: Zenodo DOI creation. Zenodo. 2017. Data Source" }	[ { "id": "30658", "date": "20 Feb 2018", "name": "Bob Siegerink", "expertise": [ "Reviewer Expertise Methodology", "epidemiology" ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThe paper by Hartgerink et al. sought to determine whether or not there is evidence for data manipulation in the DECREASE-I and Decrease-IV trials. First, the authors reproduced the results of a previous meta-analysis from Bouri et al., using raw event data to calculate the ln(relative risk) and its standard error. Next, they modeled the probability that the results from the DECREASE trials could have occurred from the effect distribution of the non-DECREASE trials. Finally, by creating simulated datasets that were subsequently manipulated they determined the probability that the likelihood of the manipulated data (DECREASE meta-analytic effect distribution) is larger than the likelihood of the genuine data (non-DECREASE meta-analytic effect distribution).\nEven though the motivating case for the paper is the questioned reliability of the data within the DECREASE trials that tries to answer a very concrete clinical question (should we use perioperatively use beta blockers to reduce mortality?), the paper ultimately tries to answer a question that is much broader: can we use forensics statistics even when individual data records are not available. In our opinion, the authors succeeded in part to answer that underlying question.\nGeneral Comments:\nSome circular reasoning in step 2, In step 2 the authors provide clear evidence that the results of the DECREASE trials are quite unlikely, however, they employ some circular reasoning in their argumentation. Because we know the DECREASE values are extreme, when a p-value is calculated for the DECREASE effect is calculated based off of the non-DECREASE effect distribution it will of course be very small. The results of this approach are methodically sound but suffer from circular reasoning and do not seem to add much to the goal of determining whether data manipulation occurred. Although this step might be useful to help ease the reader into the following steps, the limited added value of these analyses should be recognized.\n\nRobustness of approach In Step 3, the combination of adding simulated datasets with a likelihood calculation is an interesting approach to try to determine data manipulation when only summary statistics are available. In the second paragraph of the methods, when describing the inversion method, the authors say “We assumed that if data are manipulated, each data point is manipulated in the same way and to the same extent.” We have a feeling that this is a very strong assumption that is likely not to hold true and might drive the results. An exploration of the robustness, in text or analyses, could help strengthen the argument for the approach the authors apply. Additionally, since the DECREASE-IV trials have approximately 10x the sample size as DECREASE-I we expected a reduction in the confidence interval, yet the confidence interval still spans almost the entire range. Further deliberations on this phenomenon would help the reader understand the robustness of the applied technique.\n\nInterpretation of results. In general, the results show that the data simulation does not give a lot of precision when it comes to pinpointing the number of manipulated datapoints. In some sense, and as also described by the authors, this is also not needed as the lower boundary of the confidence interval of manipulated datapoints should always include 0. In fact, the authors claim that “there is a high degree of confidence that data manipulation did occur based on the difference of the trial results alone”. We do follow that reasoning, but do not fully understand why the authors adhere so much to the difference between the results of the two trials. There does not seem to be a material difference in the 95% confidence intervals between DECREASE-I and DECREASE-IV since in both cases the confidence interval spans essentially the entire range of possible outcomes. Nonetheless, the authors seem to put a lot more emphasis on the analyses of DECREASE IV with its higher sample size, in part because the results show that the lower boundary of the confidence interval does not span the 0 but ranges from 3-1066 or 10-1066, depending on the method used. Given the unknown robustness of this approach, we wonder whether the implied relevance of the small difference between the results of the DECREASE I and DECREASE IV is justified.\n\nConclusion. In the discussion section, we take issue with the sentence beginning “Our results indicate that the results from the DECREASE trials ... at least some of the data were manipulated”. The word manipulated implies an intention to manipulate data, yet there are numerous ways for data to become incorrectly classified without overt and intended manipulation. Although the authors show it is likely that something altered the data, we believe that strong claims about the provenance of that change based on the analyses provided in this paper should be avoided. However, we agree with the authors that DECREASE I and DECREASE IV should be regarded as \"highly problematic and untrustworthy when assessing the effect of beta blockade in perioperative mortality\", but that is only when we place the results from this paper in the context of the reports on scientific integrity of the DECREASE trials.\n\nNext to these general comments, we also have some specific suggestions or questions, mainly to help increase the readability in a potential second version of the manuscript :\nIt would be helpful to have a forest plot of Table 1, to better visualize the results and compare DECREASE vs. non-DECREASE. In Table 2, the authors could report the relative risk (RR) instead of the log(RR) to improve the table’s readability and help the reader understand the magnitude of the different effects depicted. We would also be interested whether some more general information on this topic, especially regarding the non-DECREASE trials to put the DECREASE trials and their data into perspective. (e.g. how many non decrease trials were registered but not published; or whether there is any other evidence or suggestion for non-publication of relevant data that could alter some of the conclusions in this paper.)\n\nA minor point, re-label the subscripts of the Likelihood function as DECREASE and non-DECREASE to avoid the loaded terminology of manipulated and genuine which seem to presuppose the conclusion.\n\nWe suggest rewording the second paragraph under the results section, and specifically address the sentence that begins “The relatively minor difference…”. This sentence first references “estimates”, but it is unclear what these estimates are referring to.\n\nIn Figure 2 please address why, in the bottom lower panel, the confidence interval for the point estimate is larger than the distribution estimate. Because the distribution estimate has more error in it, we were expecting that the confidence interval of the distribution estimate would be larger than that of the point estimate.\nThis peer review report was made in collaboration with Sean Walter Kelley, as part of his training.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] }, { "id": "34206", "date": "14 Jun 2018", "name": "Stephen Senn", "expertise": [ "Reviewer Expertise Statistical methods in drug development" ], "suggestion": "Approved", "report": "Approved\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nAn initial observation as regards my review is that I am unfamiliar with the background to this story and I have reviewed this paper only. The authors make a number of claims regarding facts that could probably be checked by reading some of the references. I have not done this. My comments are limited to the internal logic of the paper only.\nWhether or not one agrees with the conclusions, this is an interesting investigation. I have some reservations as regards emphasis in the conclusions, which I shall explain in due course, but first I shall explain why statisticians with my background may have a slightly different point of view to the authors.\nSince the time in which I worked in the pharmaceutical industry (1987-1995), I have been interested in drug regulatory science. An attitude that (rightly or wrongly) is accepted as being the norm in both sides of the regularity divide is that evidence of efficacy for one drug in a class cannot (usually) be used as evidence of efficacy of another. In fact, so strong is this point of view that when it is sought to use the proof of efficacy of one formulation for another, proof of equivalence is required. Thus, not only is it the case that different molecules cannot be regarded as having the same efficacy, this carries over to doses and formulations. Some, no doubt, will see this as a conspiracy to inhibit generic competition. However, during my own time in drug development I twice worked on alternative formulations (that is to say developed by the innovator company) that had to be abandoned. In one case a clinical trial of the new formulation revealed it had one quarter the potency of the existing formulation1.\nThis ‘no pooling of products’ attitude also holds for ‘proving’ safety. For different formulation ‘proof’ of equivalence would be required and for different molecules usually a whole new programme. (Biosimilars might be a controversial exception.) However, regulators do generally consider that observed safety problems in one molecule in a class create a potential concern. The FDA’s attitude as regards development of treatments in diabetes, partly as a response to Nissen and Wolski’s meta-analysis of rosiglitazone, is a case in point2.\nThus, there is a general aversion in drug-development and regulation, not necessarily shared by the evidence-based medicine movement, to pooling different molecules in a meta-analysis. Nevertheless, I consider that such pooling is legitimate for one purpose, namely that of testing the hypothesis that no drug in among those pooled has the effect being studied. If this is adopted as a null hypothesis and a fixed effect analysis suggests the null hypothesis should be rejected, then the hypothesis to assert becomes the hypothesis that at least one drug in the class has an effect. (See my paper on overstating evidence3 p3. ) Further investigations are then necessary to determine what the practical implications of this are, but asserting the alternative hypothesis that all drugs have an effect is clearly not warranted.\nIn fact, I can put it cynically like this. We seem to live in an era in which everyone believes in personalised medicine (thus in patient by treatment interaction) but we are unconcerned about the differences between the main effects of treatment.\nHowever, a further consequence of my time in the pharmaceutical industry, is that I share a general mistrust of investigator led-trials (with some notable exceptions). The fact that previously, raw data from pharmaceutical trials were not shared with the wider public, has been the subject of much criticism. Nevertheless, such trials were examined by the regulator and this, in my opinion, has done much to improve the quality of pharmaceutical industry trials. Without such external scrutiny the danger is that independent trials may suffer in quality.\nI now come to my specific comments.\nIt is clear from the authors’ paper that in the meta-analysis different drugs are pooled. This implies that one explanation that should not be dismissed on the basis of the statistical analysis alone, is that the effects vary from treatment to treatment in the class. To be fair to the authors, they do discuss this in more than one place but I feel that they do not appreciate fully the reservations that apply generally to pooling drugs. Again, to be fair, they do provide the extremely helpful table 2, which provides an analysis by table. It would be nice to see, just to complete the picture, if there is a connected network, what a network meta-analysis would show. (If there is not a connected network then this must be that the problem with the different experimental treatments also applies to the controls.) For example, this would permit formal comparison of different drugs. Again, it should be noted that the authors do include what might be regarded as a sort of network analysis, however, all other treatments are lumped together and this suffers from a problem discussed in point 4 below. I am not suggesting that the authors need to do this analysis (it could a be task for future work), I am just suggesting that the fact that this has not been done is a limitation that needs to be reflected in the discussion.\n\nBy the same token, this means that it would be highly debatable anyway, irrespective of any particular doubts about the reliability of the DECREASE I & IV trials, to use these as proof of efficacy of beta-blockade generally. Again, to be fair to the authors, they do suggest that more trials are needed.\n\nHowever, if the data from the DECREASE I & IV trials have not been audited, then in my opinion nobody is obliged to accept the results anyway. Checkability is the standard by which claims should be judged.\n\nI have some reservations regarding the use of the one degree of freedom chi-square tests in step 1. It should be appreciated that the analysis is one that is strictly speaking only valid if pre-specified without any access to results. It is not an analysis that is valid when comparing observed extreme results with others4.\n\nI did not understand the argument in the last paragraph of Step 3. This is probably just due to my being obtuse. The robustness of the results and the implication of the results, if true, seemed to me to get mixed up.\nHowever, these comments do not alter my opinion that this is an interesting paper.\n\nIs the work clearly and accurately presented and does it cite the current literature? Yes\n\nIs the study design appropriate and is the work technically sound? Yes\n\nAre sufficient details of methods and analysis provided to allow replication by others? Yes\n\nIf applicable, is the statistical analysis and its interpretation appropriate?\nPartly\n\nAre all the source data underlying the results available to ensure full reproducibility? Yes\n\nAre the conclusions drawn adequately supported by the results? Partly", "responses": [] } ]	1	https://f1000research.com/articles/6-1995
https://f1000research.com/articles/6-311/v1	23 Mar 17	{ "type": "Study Protocol", "title": "Scaling-up primary health care-based prevention and management of alcohol use disorder at the municipal level in middle-income countries in Latin America: Background and pre-protocol for a three-country quasi-experimental study", "authors": [ "Peter Anderson", "Amy O'Donnell", "Eileen Kaner", "Antoni Gual", "Bernd Schulte", "Augusto Pérez Gómez", "Hein de Vries", "Guillermina Natera Rey", "Jürgen Rehm", "Amy O'Donnell", "Eileen Kaner", "Antoni Gual", "Bernd Schulte", "Augusto Pérez Gómez", "Hein de Vries", "Guillermina Natera Rey", "Jürgen Rehm" ], "abstract": "Background: While primary health care (PHC)-based prevention and management of alcohol use disorder (AUD) is clinically effective and cost-effective, it remains poorly implemented in routine practice. Systematic reviews and multi-country studies have demonstrated the ability of training and support programmes to increase PHC-based screening and brief advice activity to reduce heavy drinking. However, gains have been only modest and short term at best. WHO studies have concluded that a more effective uptake could be achieved by embedding PHC activity within broader community and municipal support. Protocol: A quasi-experimental study will compare PHC-based prevention and management of AUD, operationalized by heavy drinking, in three intervention cities from Colombia, Mexico and Peru with three comparator cities from the same countries. In the implementation cities, primary health care units (PHCUs) will receive training embedded within ongoing supportive municipal action over an 18-month implementation period. In the comparator cities, practice as usual will continue at both municipal and PHCU levels. The primary outcome will be the proportion of consulting adult patients intervened with (screened and advice given to screen positives). The study is powered to detect a doubling of the outcome measure from an estimated 2.5/1,000 patients at baseline. Formal evaluation points will be at baseline, mid-point and end-point of the 18-month implementation period. We will present the ratio (plus 95% confidence interval) of the proportion of patients receiving intervention in the implementation cities with the proportions in the comparator cities. Full process evaluation will be undertaken, coupled with an analysis of potential contextual, financial and political-economy influencing factors. Discussion: This multi-country study will test the extent to which embedding PHC-based prevention and management of alcohol use disorder with supportive municipal action leads to improved scale-up of more patients with heavy drinking receiving appropriate advice and treatment.", "keywords": [ "Scale-up", "implementation", "primary health care", "cities", "alcohol use disorder", "harmful use of alcohol", "heavy drinking", "training and support" ], "content": "Introduction\n\nAlcohol use disorder (AUD) is a summary term used for the two diagnosable conditions of “harmful use of alcohol” and “alcohol dependence” within the World Health Organisation (WHO) ICD-10 classification of mental and behavioural disorders1. AUD will also be included in the ICD-11 revision under disorders due to the use of alcohol2. AUD is also a diagnosable condition within the 5th edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM–5) of the American Psychiatric Association3. AUD is associated with considerable disability, morbidity, and mortality4,5. The most recent global burden of disease analyses estimate that, worldwide in 2015, there were 63.5 million cases of AUD6 (due to more restrictive definitions, this is lower than other estimates of 95 million cases7), responsible for 137,500 deaths8, 6.3 million years lived with disability6, and 112 million disability adjusted life years9.\n\nThe WHO also uses the term ‘harmful use of alcohol’, first coined in its classification of mental and behavioural disorders in 19921, and itself a component of AUD. As a clinical term, it is a pattern of alcohol use that is causing damage to health. This will be replaced in 2017 with two clinical terms: ‘a harmful pattern of alcohol use’ (a pattern of alcohol use sustained over at least 12 months that has clinically significantly harmed the health of the user or someone else); and, ‘a single episode of harmful use of alcohol’, which has caused damage to a user’s health or someone else’s health2. Harmful use of alcohol is a term used (albeit, with different definitions) in the global NCD framework10, the WHO global strategy11, and the United Nations (UN) sustainable development goals12.\n\nMore broadly, alcohol itself is a cause of a wide range of diseases and injuries, exacerbated by occasions of heavy drinking13, resulting in it ranking as the ninth leading global risk-factor for morbidity and premature death14. Ranking increases to fourth in Colombia and Peru, and fifth in Mexico14, the three Latin American countries addressed in this protocol.\n\nAdverse impacts from AUD and the harmful use of alcohol are aggravated by lower socio-economic status15. Impacts also extend beyond the individual drinker, with considerable costs borne by families, communities, health systems, and the wider economy5. A large proportion of these costs are avertable16. Tackling the multiple individual and societal level harms caused by AUD and the harmful use of alcohol is a global economic and public health priority, and essential for achieving global targets of reducing deaths from non-communicable diseases by 25% between 2010 and 202517, more so as risk of exposure to harmful use of alcohol increases with increasing socio-economic status in low and middle income countries14. Further, UN Sustainable Development Goals Target 3.5 is to strengthen the prevention and treatment of substance abuse, including narcotic drug abuse and harmful use of alcohol, with two proposed indicators: coverage of treatment interventions (pharmacological, psychosocial and rehabilitation and aftercare services) for substance use disorders (including AUD); and, per capita alcohol consumption12.\n\nAUD and harmful use of alcohol are cumbersome definitions to use when identifying at risk patients in primary health care (PHC)18,19. An alternative is to use a level of alcohol consumption20,21. Here, the nearest definition is that used by the European Medicines Agency22, whose ‘threshold 1’ definitions of heavy drinking are more than 60g of alcohol consumed on average a day by a man and more than 40g a day by a woman. These are the same levels as original descriptions used in global burden of disease studies23. For practical purposes, we take the mid-point (50g a day) as our definition of heavy drinking. At this level of consumption, there is little difference in absolute risk (about 3.5%) of dying prematurely due to alcohol before the age of 70 years between men and women24. Hereafter, we continue to use the term heavy drinking, identified by AUDIT-C as our operational definition covering AUD and the harmful use of alcohol, for screening and advice in PHC18.\n\nDespite the fact that heavy drinking is one of the most important modifiable causes of premature morbidity and mortality25, worldwide, it is estimated that as many as four out of five heavy drinking individuals fail to receive the offer of appropriate advice or treatment26,27. In Mexico, the gap is nine out of ten28,29.The problem is not one of lack of effective treatment and prevention options30,31. A robust and extensive body of literature demonstrates the range of evidence-based strategies available to policy makers and practitioners seeking to reduce heavy drinking31,32. Questionnaire-based screening and brief advice programmes delivered in PHC are effective33 and cost-effective34 in reducing heavy drinking, even though the extent to which this evidence-base is grounded in efficacy (ideal world) or effectiveness (real world) trials is still debated in some academic circles35. In addition to brief advice, treatment for AUD and harmful alcohol use include cognitive behavioural therapy and pharmacotherapy, both of which are found to be effective in reducing heavy drinking36–39. However, to date at least, these have failed to achieve widespread up-take31,40,41.\n\nThe Organisation for Economic Co-operation and Development (OECD) has estimated that if the proportion of eligible patients receiving advice and treatment for heavy drinking increased to 30% of eligible patients, the prevalence of harmful use of alcohol could decrease by as much as 10–15% across OECD member countries, with reductions in the annual incidence of AUD of 5–14%40. Large scale implementation of advice and treatment programmes can be expensive because of staff and drug costs, but has the potential of large reductions in health care expenditures, with, in some countries, advice and treatment programmes estimated to be cost saving by large margins40. Such programmes would also free large numbers of working age people per year from alcohol-related diseases.\n\nTwo systematic reviews42,43 and two multi-country studies41,44,45 have demonstrated the possibility of increasing the proportion of patients screened, and screen-positive patients given advice by their PHC providers. The WHO Phase III four-country study on the identification and management of alcohol-related problems in primary care found that the odds ratios for the impact of training and support on increasing higher screening proportions (defined as 20% or more of eligible patients screened) was 2.2 (95% CI=1.3 to 3.1) and on increasing higher intervention proportions (defined as 10% or more of eligible patients screened and advice given to screen positives) was 2.8 (95% CI = 1.6 to 4.0), albeit from very low baseline levels45. In the more recent five-country European ODHIN (Optimizing Delivery of Health Care Interventions) study, providing training and support to PHC providers increased the number of patients screened by 50%, and providing financial reimbursement to PHC providers increased the number of patients screened by 100%, also from low baseline levels of 6/100 consulting adult patients screened41. Other evidence has suggested that the impact of financial incentives on screening and brief alcohol advice in England might have limited effects46. Although incentivised practices recorded higher levels of activity than those not paid to deliver alcohol interventions, overall rates of delivery remained low.\n\nMost work has been undertaken in high-income countries. Whilst there has been some work in low- and middle-income countries47, including countries of Latin America48–53, there is an opportunity to fast-track scale-up research and practice in such countries.54\n\nTo date, impacts in increasing PHC provider activity have been modest55. There are two important possible reasons for this, which we address in this protocol. The first reason is that standard cut-offs for the frequently used screening instrument, AUDIT-C56 (commonly five for both men and women, or five for men and four for women) to trigger advice are too low, being equivalent to an average daily alcohol consumption of about 20g of alcohol or less57. Practitioners may well find themselves averse to intervening at such low levels, which would also have huge resource implications, with one in three or four patients being eligible for advice. Cut-off points for managing raised blood pressure are commonly determined by levels of blood pressure at which treatment has shown to be effective58. Similarly, cut-off points for brief advice could be the baseline levels of alcohol consumption found in the randomized controlled trials that have investigated the effectiveness of PHC delivered brief advice. In the first Cochrane review of the topic, when reported, baseline levels ranged from 89 to 456g per week, with an overall mean across trials of 313g per week59. At a mean of 313g per week (45g per day, a little lower than the definition of heavy drinking, 50g of alcohol per day, given above), the equivalent AUDIT-C cut off would be 857. That lower cut-offs may be inappropriate is also illustrated by the lower effect sizes found in an updated Cochrane review, where the average baseline consumption at enrolment had dropped to 183g/week60. It has also been suggested that PHC providers might be more engaged in screening and giving brief advice, if screening were targeted to patients with comorbid conditions, such as depression or hypertension30,61,62. However, to date, there is insufficient evidence for an appropriate package that deals with comorbidity to scale-up63. Further, it has been shown that targeted screening misses out on the vast majority of patients that would be captured by universal screening64. Given the strong associations between harmful alcohol use and depression65,66, our protocol includes screening for depression and appropriate PHC-based management67–69 or referral for those patients identified as screen positive by AUDIT-C.\n\nThe second reason for modest increases in PHC-based activity could be due to a focus on providers alone, whereas successful implementation of health interventions within complex health system demands addressing a range of underlying structural and support systems70. Phase IV of the WHO study on the identification and management of alcohol-related problems in primary care71, outlined a range of conclusions for enhancing the widespread uptake of screening and brief advice programmes to reduce the harmful use of alcohol: (i) training and practice-based materials need local customization that can be achieved through focus groups; (ii) reframing views about alcohol of both professionals (through training) and the public (through mass media campaigns) is essential; (iii) the establishment of a lead organization is essential, gathering endorsements from a range of organisations and individuals that are highly relevant to the aims of the work; and (iv) adequately controlled community-based studies need to be undertaken to strengthen the evidence base for achieving routine implementation71. The WHO Phase IV study concluded that embedding PHC-based screening and brief advice programmes within the frame of supportive community and municipal environments might lead to improved outcomes. Experience from the US-based SAMHSA SBIRT initiative72 stressed the importance of local champions and whole practice buy in for successful implementation73,74.\n\nThis protocol outlines the design of a quasi-experimental study to test the scale-up of PHC based screening and brief advice programmes to reduce heavy drinking at city level in three Latin American middle-income countries75 (Colombia, Mexico and Peru), in which the prevalence of AUD is 6, 7 and 3%, respectively, and the prevalence of heavy episodic drinking is 4, 11 and 12%, respectively4. We will base our action on the Institute for Healthcare Improvement’s (IHI) framework for ‘going to scale’, which designates four steps in a sequence: (1) Set-up, which prepares the ground for introduction and testing of the intervention that will be taken to full scale; (2) Develop the Scalable Unit, which is an early testing phase; (3) Test of Scale-up, which then tests the intervention in a variety of settings that are likely to represent different contexts that will be encountered at full scale; and (4) Go to Full Scale, which unfolds rapidly to enable a larger number of sites or divisions to adopt and/or replicate the intervention70, see Figure 1. We call the proposed study SCALA (Scale-up of Prevention and Management of Alcohol Use Disorder in Latin America).\n\nThe four phases of going to scale from setting up the programme within the three cities to exploiting the validated framework and strategy through city networks, with the adoption mechanisms and support systems. PHC, primary health care.\n\nDriven by implementation science70,76–83, this three-country study aims to test the extent to which embedding PHC-based screening and brief advice activity within supportive municipal action leads to improved scale-up of more patients with heavy drinking receiving appropriate advice and treatment. The study has the following objectives:\n\n1. To deliver a tailored package for improving prevention and early identification of heavy drinking, with advice and treatment for case positives that is scalable at municipal level in a wide range of middle- income countries;\n\n2. To set-up and implement the scalable package with key stakeholders in three case study cities (scalable units) from Colombia, Mexico and Peru;\n\n3. To test the scale-up of the package for its impact on provider delivery of early identification and management, and on patient-level alcohol consumption and satisfaction;\n\n4. To identify and document the facilitators and barriers, and the organizational and resource requirements for going to full-scale, including full economic analyses; and\n\n5. To present a validated framework and strategy for going to full-scale, embedding the package into routine policy and practice, taking into account aspects of stigmatization and equity, that can be replicated globally in the future throughout municipalities.\n\nCountries from Latin America are selected as this is a sub-region of the world in which alcohol jumps from ninth globally to the fourth most important risk factor for morbidity and premature death14. The three specific middle-income countries are chosen to represent Central (Colombia and Mexico) and Andean (Peru) Latin America. The three countries have pre-existing collaboration between the authors, who have experience in the area48–53.\n\n\nProtocol\n\nThe study is a quasi-experimental design84, comparing changes in screening and brief advice, and, if relevant, referral for treatment activity, amongst primary health care units (PHCUs) in intervention cities with PHCUs in similar control cities, Figure 2.\n\nPHCU, primary health care unit.\n\nCities will be selected from Colombia, Mexico and Peru. Criteria for choice of scale-up city will include existing links with one or more authors, willingness of the city to be involved, and ability to recruit PHCUs. Comparator cities will be chosen on the basis of comparability with the scale-up city in terms of wealth and other characteristics which impact on drinking, health care and survival, and geographical location to minimize spill over effects from the scale-up city. Cities are chosen as the scalable unit, as there is a systemic global trend for municipalities to increasingly take on the jurisdictional responsibilities for prevention and health care services. Cities, themselves, are active in prevention and health promotion programmes, and there is a strong evidence base for their impact, also in the prevention of alcohol-related harm85,86. Cities are a natural site for preventing alcohol-related harm87. Although not having the full jurisdictional responsibilities of national governments for all alcohol policy issues, they often have greater flexibility and are an important site for both media-based and social norms programmes, as well as environmental measures to manage and limit availability of alcohol88. Networks of cities are natural vehicles for exploitation of the results and deployment to full scale, more so with the trends of increasing urbanization in Latin America89,90.\n\nPrimary care-focused health initiatives can improve access to health care, including among the poor, at reasonably low cost in low- and middle-income countries91, and particularly so in Latin America90. Health-system reforms in Latin America have placed a strong emphasis on the development of comprehensive PHC as a vehicle to achieve universal health coverage, reduce inequities, and democratise health through participation. However, they face ongoing challenges, in particular, the development of health services that can meet the emerging health needs brought on by social and demographic transitions, including the increasing chronic disease burden, and the impacts of rapid urbanisation92.\n\nManagement of chronic diseases relies on opportunistic case finding, assessment of risk factors, detection of early disease, identification of high-risk status, combined psychosocial and pharmacological interventions, and long-term follow-up with regular monitoring and promotion of adherence to advice and treatment. Such approaches are financially feasible and have the potential to substantially reduce the burden of chronic diseases. Many interventions can be managed effectively by non-specialists and lay health care workers who are supported by specialists. Although implemented in a range of settings, collaborative care models seem best delivered in PHC settings93. Evidence demonstrates the effectiveness of PHC-based lifestyle interventions in Latin American contexts94,95, including brief advice programmes to reduce heavy drinking, as well as the potential to detect and refer high-risk patients.\n\nApproximately ten PHCUs per intervention and comparator cities will be involved, 60 PHCUs in total. The exact number of PHCUs will depend on the average number of registered patients per PHCU. In each city, the total number of recruited PHCUs should cover a population of about 80,000 registered patients (including children and adults). In jurisdictions, where PHC physicians work as individual practitioners, a PHCU can be defined for the purposes of the study as a virtual or physical location where three or more PHC physicians work. Identification of PHCUs who agree to participate in the studies will be drawn from administrative or academic registries of PHCUs at national, regional, or city levels. The process of recruiting PHCU will be described in detail by each country. Within each PHCU, eligible providers will include any fully trained medical practitioner, nurse or practice assistant with a non-temporary employment contract, working in the PHCU and involved in medical and/or preventive care. These providers will sign an informed consent for their participation. Dependent on customary country practice, participating PHCUs will receive a study fee.\n\nThe SCALA care pathway includes three integrated components:\n\ni. preventing the development of heavy drinking via increased alcohol health literacy;\n\nii. screening and brief advice to reduce the prevalence of heavy drinking; and\n\niii. diagnosis and clinical management of severe AUD and/or co-morbid depression.\n\nThe SCALA intervention package deals primarily with the first two parts, prevention and management of heavy drinking. It does not specifically address managing severe AUD, including alcohol-related physical complications and/or severe co-morbid mental health conditions, but ensures the necessary links with specialist services in order to do so, even though specialist treatment can be managed in PHC, with appropriate support67,96.\n\nWhilst AUDIT-C is highly effective at identifying heavy drinking, it is not designed to stratify patients by severity of AUD, nor designed to diagnose depression, commonly comorbid with heavy drinking. A DSM-5 11-item instrument can be used to stratify the severity of AUD into mild (2–3 items), moderate (4–5 items) and severe (6+ items)97. Similarly, the Patient Health Questionnaire 9 (PHQ-9), can be used to diagnose moderately severe or severe depression with a cut-off score of 15+98. In our protocol, patients scoring 8+ on AUDIT-C, will be further screened with the DSM-5 11-item instrument and the PHQ-9 to assess severity of AUD and to identify patients with co-morbid depression.\n\nFor the care pathway (Figure 3), all adult patients (age 18+ years) visiting the PHCU for whatever reason will be screened with AUDIT-C, with country-specific pictograms of standard alcohol beverages used to identify the standard unit (drink) of alcohol. Patients with an AUDIT-C score of <8 will be given a patient information leaflet to improve alcohol health literacy (knowledge of the risks of drinking alcohol, and skills to achieve and maintain lower risk drinking, defined as no more than 20g of alcohol per day). Patients with an AUDIT-C score of 8+ will be invited to complete the DSM-5 11-item instrument and the PHQ-9: those with an 11-item score of <6 and a PHQ-9 score of < 15 will be given brief advice of between 5-10 minutes, based on the FRAMES principles99. Those with an 11-item score of 6+ and/or a PHQ-9 score of 15+ will be refereed to more specialist services, at the clinical decision of the health care provider A record of what steps are taken will be recorded on paper or electronic tally sheets prepared for the study.\n\nFor screen negative patients, screen positive patients without AUD and depression and for screen positive patients with AUD and/or depression. PHCU, primary health care unit; AUD, alcohol use disorder.\n\nIn the intervention cities, implementation strategies will comprise three components: tailoring the PHC screening and advice package; providing specific practice-based training and ongoing support to PHCUs; and, implementing city-based adoption mechanisms and support systems, including media-based campaigns to improve alcohol health literacy. In the intervention cities, all PHCUs will be given a summary card of screening and advice procedures, with instruction, instruction on how to complete record sheets, and record sheets. In the control cities, all PHCUs will be given a summary card of screening and advice protocol, with no instruction, instruction on how to complete record sheets, and record sheets. As part of the study, no other action will take place in the control cities.\n\nSCALA is a trans-cultural study, with different health systems, and differences in drinking patterns and attendance at PHC centres, compounded by gender differences. In Mexico, for example, men consume more alcohol then women, but attend PHC services much less frequently than women53. Thus, there is a need for careful tailoring of the screening and advice package. Each intervention city will create Community Advisory Boards (CABs) representing academia, city health and public health departments, health service commissioners and practitioners, and patient and public engagement groups; and User Panels (UPs) of user groups, including PHC providers, patients and citizens. Through expert meetings, workshops, and focus groups, the package will be fine-tuned and tailored to the needs of each city, based on the Tailored Implementation for Chronic Diseases initiative100–102 within the seven domains of: local and national guideline factors; individual health care provider factors; patient factors; interactions between different professional groups; incentives and resources; capacity for organizational change; and, social, political and legal factors. At the city level, tailoring will be based on the principles of integration between PHC and municipal services103 and the development of complementary community ecosystems that support reductions in heavy drinking. At the PHC level, tailoring will be based on the principles of co-production of health104 between PHC providers and patients.\n\nIn the intervention cities, PHCUs will be offered two initial two-hour face-to-face educational trainings prior to the 18-month scale-up phase, and two one-hour booster sessions during the first twelve months of the 18-month scale-up phase. Training will take place within the PHCU or clusters of PHCUs. Training will be undertaken by peer trainers, members of the research team, accredited teachers, or addiction consultants. Training will focus on practical skills in undertaking screening and in delivering brief advice, in using the questionnaires, and in knowing when and how to refer patients with more severe AUD105–108 and moderately severe or severe depression67. Training will, in addition, address attitudes, and perceived barriers and facilitators109–111 in implementing screening and brief advice, contextualized to local circumstances112. Each country will use an adapted existing country-based training and support package. Where these do not exist, training and support packages will be adapted based on the PHEPA (Primary Health Care European Project on Alcohol) training programme113, widely implemented since 2002 in Catalonia, a Catalan/Spanish speaking, bilingual geographic area. The PHEPA training programme is similar to those used in the WHO Phase III trial45 and the ODHIN study41.\n\nWithin each intervention city, an integrator (champion and knowledge and practice broker) will be appointed with responsibilities of serving as a trusted and accountable leader: facilitating agreement within the city and health systems on shared goals and metrics; assessing and acting on relevant community resources; working at the systems level to make relevant practice changes for sustainability; gathering, analyzing, monitoring, integrating, learning, and sharing data at the individual PHCU and city levels; identifying and connecting with system navigators who help PHCUs coordinate, access, and manage multiple services and supports; and developing a system of ongoing and intentional communication with PHCUs and cities.\n\nFive adoption mechanisms will be used for scale-up: (i) demonstration of the superiority of the PHC package, its simplicity, and its alignment with the latest evidence of preventing and managing heavy drinking and of implementation science; (ii) engagement of identified leaders and building their capacity to lead and ensure broad adoption of the PHC package through guiding and supporting large-scale change114–116; (iii) communicating the value of the PHC package to both municipal and PHC frontline staff117; (iv) identifying and adjusting, as appropriate and possible, relevant policies at PHC and city levels to expedite the adoption of the PHC package; and, (v) identifying gaps in health system performance and the urgent need to prevent and manage heavy drinking to promote the needed will and energy to bring implementation of the PHC package to scale118. An additional five mechanisms will be used to support scale-up: (i) development of professional capacity for scale-up; (ii) development of infrastructure for scale-up, achieved through redesign rather than addition of new resources; (iii) linking to monitoring and evaluation, using reliable data collection and reporting systems that track and provide feedback on the performance of key processes and outcomes, for example monthly reporting on screening and brief advice activity; (iv) setting up learning systems to capture change ideas that are shown to result in improved performance assembling ideas into a change package. Knowledge should be shared between municipal actors and PHCUs through regular electronic newsletters and communications119; and, (v) creating design factors that enhance sustainability including high reliability of the new processes, inspection systems to ensure desired results are being achieved, support for structural elements, and ongoing learning systems120,121.\n\nBased on the validated methodology of the ODHIN project41,122, PHC providers will document activity by completing paper or electronic (depending on the health system, and the ability to use existing electronic health records) anonymous tally sheets that record eligible patients’ (aged 18+ years) AUDIT-C scores, if administered, DSM-5 11-item and PHQ-9 scores, and the advice or treatment given to each patient. The tally sheets will record the age, sex, employment status, and educational level of the patient, the latter as one proxy measure of socio-economic status. The tally sheets will also include: two questions that capture previous experience of being asked about how much the patient drinks and of being advised to reduce the amount drunk to provide information for UN Sustainable Development Goal 3.512; one question about alcohol being a cause of high blood pressure, liver problems, depression or cancer, as a simple measure of alcohol health literacy (knowledge part)123; and, two questions about injunctive social norms of drinking alcohol124.\n\nData will be collected for each calendar month during the 18-month scale-up period. Formal evaluation will take place during three measurement periods: 4-week baseline period; 4-week assessment period during the 9th month of the 18-month scale-up period; and, 4-week assessment period at 18-months, the end of scale-up period. PHCUs will return data on the number of adult (aged 18+ years) consultations per provider for the four-week baseline assessment period, and for each of the 18 months of the scale-up period.\n\nAt baseline, PHC providers will provide data on their age, sex and profession (doctor, nurse, practice assistant etc.). At baseline, and at two time points during the 18-month scale-up period (month 4.5 and month 13.5), providers will provide data on their alcohol health literacy and on their attitudes to working with patients with heavy drinking. The alcohol health literacy instrument will assess knowledge of risks due to drinking123, and descriptive and injunctive social norms124. The attitudes instrument will be the shortened version of the Alcohol and Alcohol Problems Perception questionnaire125.\n\nDuring month 3 of the 18-month implementation period, the first six consecutive screen positive patients identified by each PHC provider will be invited to give their consent to complete two follow-up questionnaires, at six months and twelve months after the initial screening. The patient interviews will be used for quality control126, but not as a study outcome measure. The follow-up questionnaires will be the same as the baseline questionnaire and will be undertaken by the local academic unit by face-to-face or telephone interview. Collected data will include sex, age, educational level, alcohol consumption (operationalized by AUDIT-C), alcohol health literacy, prevalence of depressive symptoms using the nine-item patient health questionnaire98, experience of screening and brief advice and treatment for heavy drinking, experience of self- and co-management for heavy drinking and health service utilization.\n\nProcess evaluation will be ongoing through interviews with CABs, with formal evaluation time points at baseline, ninth month of the 18-month scale-up period, and at the end of the scale-up period. Logic models will be developed and data will be collected on drivers, facilitators and barriers of successful implementation127,128. City and country-based contextual, financial and political-economy factors will be collected (see outcomes below).\n\nDuring all phases of the scale-up, we will document impact on other sectors (education, social care, criminal and justice, etc.) based on resource use measurement129. Patients in the scale-up and comparator cities will be asked to complete a short questionnaire about resource use measurement. Costs will be calculated by multiplying volumes (resource use) with unit costs, based on guideline prices130. Quality Adjusted Life Years (QALYs) will be measured by the local version of EuroQol131, a self-administered questionnaire, which measures both generic quality of life, as well as utilities. It contains five dimensions of health-related quality of life, namely mobility, self-care, daily activities, pain/discomfort and depression/anxiety, that can be summed into a health state, with calculable utility values132. The utility values will be used to compute QALYs. A probabilistic Markov decision analytic model will be built in to estimate the expected cost per outcome and the costs per QALY of SCALA from a societal perspective, based on established economic evaluation state-transition modelling guidelines133,134. Costs and effects will be modelled for five years and life time. Probabilistic sensitivity analyses will be executed.\n\nAll relevant data required for testing the scale-up will be transferred to the institution leading the evaluation work (Technische Universitaet Dresden) in accordance with its research data protocols. No individual data will be published, and data will only appear in aggregate form in project publications. On publication of the results, datasets will be made available via the UK data archive service (http://www.data-archive.ac.uk/).\n\nPrimary outcome: The primary outcome will be the proportion of consulting adult patients intervened (screened and advice given to screen positives), calculated as the number of AUDIT-C positive patients that received oral advice or referral for advice to another provider in or outside the PHCU, divided by the total number of adult consultations of the participating providers per provider and per PHCU.\n\nSecondary outcomes:\n\n- Screening and advice: The proportion of patients screened will be calculated as the number of completed screens divided by the total number of consultations of all patients eligible for screening (as defined above) per participating provider, and averaged per participating PHCU. The proportion of patients advised will be calculated as the number of brief interventions delivered (received oral brief advice, and/or were given an advice leaflet, and/or were referred to another provider in or outside the practice), divided by the total number of screen positives per participating provider and averaged per participating PHCU. Information will also be collected on the number of screen negatives who received brief advice.\n\n- Provider attitudes and provider alcohol health literacy: Attitudes of the participating providers will be measured by the short version of the Alcohol and Alcohol Problems Perception questionnaire, SAAPPQ25,135–137. The responses will be summed within the two scales of role security and therapeutic commitment. Individual missing values for any of the items in a domain will be assigned the mean value of the remaining items of the domain before summation. Provider alcohol health literacy will be assessed through knowledge of risks due to drinking123, and reported descriptive and injunctive social norms of drinking124.\n\nWe will use the RE-AIM Framework as our basis to evaluate SCALA’s impact across the five dimensions of reach, efficacy, adoption, implementation, and maintenance138–140, ensuring fidelity in its completion141, Figure 4.\n\nPHCU, primary health care unit; PHC, primary health care; AUD, alcohol use disorder.\n\nAt least four elements will be included. First, a driver diagram142 will be used to identify drivers for successful scale-up. To enable a nuanced understanding of how scale-up varies in the different cities, recognizing that context can have a greater influence on scale-up than any pre-specified implementation strategy, the driver diagrams will provide real- time continuous feedback on how changing contexts in health systems or city actions affect outcomes. Second, the evaluation procedure of WHO’s Urban Health Equity Assessment and Response Tool (Urban HEART)143 will be modified to identify the barriers and facilitators to scale-up. Third, the factors influencing the progress from scale-up to outcomes will be identified and documented based on UK Medical Research Council guidance144 analysing factors within five groups: (i) description of intervention and its causal assumptions; (ii) context; (iii) implementation; (iv) mechanisms of impact; and, (v) outcomes. Fourth, using the detailed methodology of Ysa et al. 145, the experience and outcomes of the scale-up will be mapped with contextual, financial and political-economy analyses of the cities and the countries within which they are located. The following contextual factors will be collected: (1) available data similar to that of the OECD better life initiative146, including material living conditions (housing, income and jobs) and quality of life (community, education, environment, governance, health, life satisfaction, safety and work-life balance); (2) Sustainable Governance Indicators147, including the Status Index, which ‘examines each state’s reform needs in terms of the quality of democracy and performance in key policy fields’, and the Management Index, focused on ‘governance capacities in terms of steering capability and accountability’; and, (3) World Values Survey data148,149 for cross-cultural variation (Traditional vs. Secular-rational; and, Survival vs. Self-expression). Documentation will be complied either at municipal or country level for alcohol policy-related strategies, action plans, legislation and evaluations. A model will be built on two levels of analyses, contextual factors and policy factors and this will be mapped on to the test of the scale-up of the PHC interventions to describe and identify those contextual and policy factors that might influence going to full-scale beyond the implementation cities.\n\nOur power calculations are based on the following assumptions: at baseline, 2.5/1,000 consulting patients will be found to be screen positive (based on an AUDIT-C cut-off score of 8) and advised to reduce their alcohol consumption (data from ODHIN study; Anderson, personal communication). To detect an increase in the number to 5/1,000 (a doubling), with 80% power and a significance level of 5%, and assuming a design effect of ten PHCUs per three cities per group (scale-up and comparator), with an ICC for PHCUs across countries = 0.03 (data from ODHIN study; Anderson, personal communication), a conservative estimate of 30 PHCUs across three scale-up cities and 30 across three comparator cities, about ten per city will be needed150, assuming an average PHCU size of about 8,000 patients with a monthly consultation rate of 1,200 adult patients per PHCU (data from ODHIN study; Anderson, personal communication).\n\nThe primary outcome of the study will be the proportion of consulting adult patients intervened (screened and advice given to screen positives) measured during two four-week periods midway and at the end of the 18-month scale-up period, and this will be analysed at the levels of the PHCU and provider by city type (intervention or control)151. Given the rarity of the event and the resulting distribution, we will use exact inference methods for comparison of intervention vs. control cities. For further analyses, including covariates, regression models will be used, taking into consideration the hierarchical nature of the data152, and characteristics at different hierarchy levels (i.e., characteristics of the PHCU, characteristics at the city level, such as patterns of drinking), and incorporating 4-week baseline period measurements as covariates. Special consideration will be given to the skewness of data by applying models, such as zero-inflated binomial regression, after testing for necessary assumptions153,154. Odds ratios will be presented with 95% confidence intervals. For any PHCU or provider that drops out during the study, outcome values for subsequent measurement points will be set at the last value obtained.\n\nBefore any involvement of participants in the study, including patients consulting in the study PHC units, the respective country-based partner in Colombia, Mexico and Peru will comply with their national legislation, regulations and ethical principles by applying for an ethical approval for research at the competent ethical authorities in their jurisdiction.\n\n\nDiscussion\n\nThis protocol outlines a quasi-experimental study84 to test the extent to which embedding PHC-based screening and brief advice activity within supportive municipal action leads to improved scale-up of more patients with heavy drinking receiving appropriate advice and treatment. It uses the IHI framework for going to scale70 to address the well-known evidence to practice gap of screening and brief advice for heavy drinking in PHC.\n\nThe proposed study has several features than merit attention.\n\nFirst, we simplify and account for cultural differences in definitions of AUD18,19, by using heavy drinking20,21 as our operational approach, rather than AUD or harmful use of alcohol1–3.\n\nSecond, we set a higher cut-off score for AUDIT-C (8+) than is commonly used to classify screened case-positives, matching definitions of heavy drinking23,24, and similar to baseline levels of alcohol consumption in PHC-based trials to reduce heavy drinking59. We also set the same cut-offs for men and women, based on epidemiological evidence24, and minimizing unintended consequences of using different cut offs for men and women155.\n\nThird, we limit brief advice to 5-10 minutes, rather than using more intensive interventions67, since the evidence suggests that brief advice is as effective and cost-effective as more extended advice or treatment in reducing heavy drinking33,34,156,157.\n\nFourth, we recognize the importance of comorbid moderately severe and severe depression65,66, by building in identification and referral mechanisms, recognizing that moderately severe and severe depression can be well-managed with sufficient support systems in PHC67–69.\n\nFifth, based on evidence71, we adopt a novel approach by embedding and scaling-up the PHC activity within cities, supported by a series of city-based adoption mechanisms and support systems70, and enhanced alcohol health literacy158, aiming to assist in building a new knowledge base, on which better policy could be based.\n\nSixth, we use a theory-based approach to tailoring100–102, creating city-based Community Advisory Boards, and user-based UPs to ensure that tailoring matches user needs, municipal services103, and co-production of health93,104.\n\nSeventh, we include a range of outcome measures, including patient outcomes, as a quality check126, which address weaknesses of many previous implementation studies in this area, which have focussed on provider outcomes, rather than patient outcomes42–43.\n\nEighth, we have a longer time frame (18 months) than is traditionally used in implementation studies41,44,45, to assess longer term impacts.\n\nNinth, we give considerable emphasis to process evaluation144, developing logic models to document the fidelity of all implementation strategies, and to identify, the drivers and barriers and facilitators to successful implementation and scale-up, and the political and economic contextual factors that might influence scale-up, based on the RE-AIM framework138.\n\nAnd, finally, tenth, we place the study design in the public domain, so that others might replicate the study approach (with acknowledgment) to see if the scale-up principles can work across jurisdictions. In so doing, we would be pleased to receive comment and feedback.\n\nWe are aware of some limitations of the study design. As we are unable to randomize the involved cities, we are unable to conduct a randomized controlled trial. Our design is rather a quasi-experimental design. The studies are approached pragmatically. In other words, each of the involved countries differs slightly in the implementation strategy contents. For example, countries will differ in their distribution of research fees and delivery of training and support strategies. The participating countries differ in their organization of PHC services and have different drinking patterns. However, this also creates opportunities to conduct across country analyses and relate different outcomes to cultural and organizational differences. The results can consequently be applicable throughout a range of countries. Although our focus on embedding PHC activity within supportive municipal actions is hypothesized to increase screening and brief activity over and above that previously demonstrated, such an approach also brings risks. Municipal governments change; and, thus health priorities may change. Although our approach minimizes the need for extra resources (and in some jurisdictions, could be resource saving34,40), it is not resource free. Funding constraints could limit future scale-up and sustainability.", "appendix": "Author contributions\n\n\n\nPA, AO’D, EK and JR conceived the study. JR undertook the power calculations and wrote the statistical measures. PA drafted the manuscript. AO’D, EK, AG, BS, APG, HdV, GNR and JR revised the manuscript at all stages of preparation and approved the final version.\n\n\nCompeting interests\n\n\n\nNo competing interests were declared.\n\n\nGrant information\n\nThe author(s) declared that no grants were involved in supporting this work.\n\n\nAcknowledgments\n\nAn application for funding for the study from the European Commission is currently under preparation.\n\n\nReferences\n\nWorld Health Organization (ICD-10). Reference Source\n\nWorld Health Organization (ICD-11). Reference Source\n\nAmerican Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders. Washington, DC, American Psychiatric Association. 2013. Reference Source\n\nWorld Health Organization: Global Status Report on Alcohol and Health 2014. Geneva, Switzerland: WHO Press. 2014. Reference Source\n\nRehm J, Mathers C, Popova S, et al.: Global burden of disease and injury and economic cost attributable to alcohol use and alcohol-use disorders. 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PubMed Abstract \| Publisher Full Text \| Free Full Text" }	[ { "id": "22773", "date": "23 Jun 2017", "name": "John B. Saunders", "expertise": [ "Reviewer Expertise Screening and brief interventions", "diagnostic concepts and guidelines", "susceptibility to alcohol- and drug-related disorders", "physical sequelae such as liver and circulatory disease", "new addictive disorders (such as gaming disorder)", "treatment of alcohol", "opioid and psychostimulant disorders", "and medical education." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis paper, which is in the nature of a protocol paper, describes an ambitious three country (six city, 60 primary health care centre) study in Central and South America which seeks to compare the response to the implementation of a comprehensive support strategy for an alcohol screening and brief management approach (and also screening for depression) in comparison with a no support condition, adopting a quasi-experimental design for the comparison. The authors claim that this is the first such controlled study of this type based in middle-income countries. No results of pilot work in these countries are presented but the authors draw upon comparable work undertaken in Europe and the US.\n\nSpecific Comments\n\nThe following are points for the authors to consider:\n\nThe introduction starts awkwardly with a description of the alternative ways of conceptualising alcohol misuse. Some of the later statements contradict what is stated earlier about the relationship between alcohol use disorder, alcohol dependence, harmful alcohol use, and risky drinking. What is needed particularly in a scale-up of alcohol screening and intervention is a simple conceptual model, and these exist. There is a natural hierarchy of (1) hazardous/risky drinking (i.e. without harm), (2) harmful drinking (i.e. with harm), and (3) alcohol dependence (where there is a psychobiological driving force to drink not seen in (1) or (2)). Some term the whole spectrum “alcohol misuse” and others “unhealthy alcohol use.” Indeed, such a composite entity was used as the reference standard against which the original AUDIT was gauged. For the definitive version of the paper, I would strongly suggest that the relevant sections of the introduction be reshaped.\n\nWith regard to the “treatment gap” caution is advised about extrapolating too much from the findings of persons who have treatment to those who have or might not. The latter, although fulfilling the relevant diagnostic criteria, have less severe alcohol misuse and less psychiatric comorbidity and are less complex than those who get into treatment.\n\nIs there any work reported for the three countries selected on the professional supports available to PHC staff? These might include (1) clinical supervision, (2) specialist to generalist support – mentioned in the paper but I can’t see what this would mean - ? on site, by telephone, smartphone apps, (3) peer support – on site or city groupings, (4) administrative support. Is it known what PHC staff would like as an incentive to undertake this work?\n\nReference is made to various scaling-up approaches but the paper would be strengthened by a description and discussion of the theoretical concepts underlying the framework proposed. How does the four-point framework relate to established implementation approaches, which help move organisations into the “early adopter” category? How do the principles of competitive advantage apply, because there will likely be competing proposals for PHC involvement in these countries e.g. childhood immunisation? The use of local “champions” is appropriate but what is the conceptual and empirical basis for this approach? Note that these comments do not refer to the IHI scale-up approach, which is about process.\n\nAlcohol screening and intervention outputs will be assessed by the AUDIT-C questionnaires completed and annotated. Depression screening (with the PHQ-9) and intervention is much less emphasised in the body of the paper. Do all the components of the implementation strategy apply equally to this? Will the investigators have access to medical records and PHC throughput data?\n\nOverall, this is a very ambitious and complex research program and I became concerned in reading it - in all its complexity - that it might not be achievable with staff who have no prior involvement in research and who are working in resource-constrained health care systems. For example, there is a statement “During all phases of the scale-up, we will document impact on other sectors (education, social care, criminal and justice, etc.) base on resource use measurement.” This is a huge additional amount of work.\n\nThe paper would be more convincing (to this reviewer) if it were linked to budgetary inputs. I appreciate that the authors are seeking European Union funding. If this becomes available, the practicalities of doing this study could be set out much more clearly.\n\nAlthough there are approximately 160 references, an impressive number for a protocol paper, there are some surprising omissions, including work which has been specifically addressing implementation of alcohol interventions and including papers that were published from phases of the World Health Organization Implementation Studies and of which the authors of the present paper are also authors. An example is: Funk M, Wutzke S, Kaner E, Anderson P, Pas L, McCormick R, Gual A, Barfod S, Saunders JB. A multi-country controlled trial of strategies to promote dissemination and implementation of brief alcohol intervention in primary health care: Findings of a World Health Organization Collaborative Study. Journal of Studies on Alcohol 2005; 66: 379-388.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Yes\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "2908", "date": "28 Jul 2017", "name": "Peter Anderson", "role": "Author Response", "response": "The introduction starts awkwardly with a description of the alternative ways of conceptualising alcohol misuse. For the definitive version of the paper, I would strongly suggest that the relevant sections of the introduction be reshaped.The nomenclature describing ‘harmful use of alcohol’ and ‘alcohol use disorders’ are cumbersome and somewhat contradictory across organizations and publications, with, for example, three distinct and contradictory concepts of “harmful use” in WHO documents of the last five years alone. As mentioned and referenced in the paper, several of us have argued for, and have published for, clarity, arguing that the term ‘heavy use over time’ can be a simplified replacement descriptor. This ties in well with the epidemiology and simplifies approaches for primary health care clinicians. It fits well with the use of AUDIT-C as the screening instrument, where the overwhelming majority of the variance is due to the consumption items. It is similar to models managing blood pressure. We and the reviewer will probably not come to agreement on this approach. We have carefully re-read the introduction and consider that it accurately reflects the current usages of the terms alcohol use disorder and harmful use of alcohol as used by various institutions and reference bodies. We consider heavy drinking a more appropriate term as the simple conceptual model for use in primary health care, with cut-offs for intervention based on levels at which advice has been shown to be effective (similar to managing blood pressure, as we argue in the section overcoming constraints on PHC activity). The introduction as written reflects the approach and concepts that we are taking in the protocol, and, thus, we do not consider that it is appropriate to adjust. With regard to the “treatment gap” caution is advised about extrapolating too much from the findings Yes, this is likely true. Nevertheless, there is an apparent and widespread gap between those who might benefit from advice and treatment and those who get it (like high blood pressure). We have added some words of caution in measuring and reporting treatment gaps. Is there any work reported for the three countries selected on the professional supports available to PHC staff? There have been existing studies on testing the impact and implementation of primary health care based screening and advice programmes in Latin American countries, and these are referenced in the paper. We describe the training and support and the city-based adoption and support systems in the paper. We have revised this a little to reflect the reviewer comments. Our collective experience is that many primary health care centres are willing to undertake this work, as they understand the importance of it. Nevertheless, in the project budget, small research fees are allocated for the primary health care centres. If the strategy is proven to work, the expectation is that Departments of Health will adopt the approach as municipal/national strategies. Reference is made to various scaling-up approaches but the paper would be strengthened by a description and discussion of the theoretical concepts underlying the framework proposed. We have added text on implementation and scale-up literature to the first part of the discussion. Will the investigators have access to medical records and PHC throughput data?In all three countries, electronic health records are used. Provided ethical and confidentiality agreements are adhered to, anonymous data can be extracted allowing, through confidential identification systems, linkages between questionnaire scores, primary health care consultations and hospitalizations by diagnostic codes. We have added a sentence about this in the discussion. Overall, this is a very ambitious and complex research program and I became concerned in reading it - in all its complexity - that it might not be achievable with staff who have no prior involvement in research and who are working in resource-constrained health care systems.We respond by considering that these are false and prejudicial assumptions. The CVs and experiences of the investigators in the three country sites surpass those of many investigators who have implemented similar international studies in ‘high-income’ countries. Further, in the experience of many of the authors of this paper, innovation in implementing screening and brief advice programmes in many Latin American countries surpass that of many high-income countries. Coupled with extensive use of leading technologies, including linked electronic health records, health systems in Latin American countries, whilst relatively resource constrained, are sometimes far ahead than health systems in many ‘high income’ countries. There is a statement “During all phases of the scale-up, we will document impact on other sectors (education, social care, criminal and justice, etc.) base on resource use measurement.” This is a huge additional amount of work.This is a misunderstanding. It requires quite minimal data collection from samples of patients. There are then robust tried and tested methodologies to estimate costs per outcome based on the collected data. In any case, these estimates are a requirement within the call for proposals. The paper would be more convincing (to this reviewer) if it were linked to budgetary inputs. The purpose of a protocol is to describe the scientific approach. We would not be proposing this study if we did not consider that we had sufficient resources to implement it. Although there are approximately 160 references, an impressive number for a protocol paper, there are some surprising omissionsWe have included references to the WHO Phase III and IV studies, which the first author of this paper coordinated whilst he worked with WHO. We have added the Funk et al. reference (REF 159)." } ] }, { "id": "23258", "date": "26 Jun 2017", "name": "Peter Nygaard", "expertise": [ "Reviewer Expertise I have conducted research on obstacles with implementation of SBI in primary health care", "and I am familiar with the literature in this field." ], "suggestion": "Approved With Reservations", "report": "Approved With Reservations\n\ninfo_outline\nAlongside their report, reviewers assign a status to the article:\n\nApproved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested\n\nApproved with reservations\nA number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.\n\nNot approved Fundamental flaws in the paper seriously undermine the findings and conclusions\n\nThis is a very important and ambitious study that focuses on some of the known obstacles of implementing screening and brief intervention in primary health care (PHC). However, whereas some of the inherent problems in the procedures are being tested, others remain untouched. For example, it is quite unclear how the authors intend to maintain the interventions after the project ends. It is well-known that you can bring GPs to test SBI interventions but once the projects are over, they return to practice as before. Below are a few points that came to mind reading the article.\nIt is quite unclear what the focus of this study is because the primary outcome is basically a rise in patients screened and given advice. It should be evident that this number will go up in participating PHCs because of the intervention per se???\nThe process evaluation is definitely a strength of this study because it is comprehensive with a very clear framework of understanding.\nIt is difficult to see the advantage of involving three different countries with only one intervention city and one control city from each. As pointed out, there are differences among the participating countries that will have to be accounted for and that will limit the robustness of the results. Furthermore, it is unclear how the participating cities will be chosen and how the intervention city will be chosen. Including three different “cultures” with only one intervention and one control site for each does not seem to be a robust design.\nThere should be more considerations about the differences among the participating countries and the impact on the final outcomes. This is a very important study that focuses on some of the known obstacles of implementing screening and brief intervention in primary health care (PHC). However, whereas some of the inherent problems in the procedures are being tested, others remain untouched. For example, it is quite unclear how the authors intend to maintain the interventions after the project ends. It is well-known that you can bring GPs to test SBI interventions but once the projects are over, they return to practice as before.\n\nIs the rationale for, and objectives of, the study clearly described? Yes\n\nIs the study design appropriate for the research question? Partly\n\nAre sufficient details of the methods provided to allow replication by others? Partly\n\nAre the datasets clearly presented in a useable and accessible format? No", "responses": [ { "c_id": "2907", "date": "28 Jul 2017", "name": "Peter Anderson", "role": "Author Response", "response": "It is quite unclear how the authors intend to maintain the interventions after the project ends. The last major international study in this area, the Phase IV WHO study, referenced in the paper, concluded that embedding primary health care-based screening and brief advice programmes within the frame of supportive community and municipal environments might lead to improved outcomes, and called for adequately controlled community-based studies to be undertaken to strengthen the evidence base for achieving routine implementation. The present study aims to do that - we have clarified this by adding a hypothesis. It is not completely correct to say that once projects are over, GPs return to practice as before. In the ODHIN study, six months after the completion of the implementation period and the brief training received, providers who had received training were still screening and advising more patients than those who had not received training. It is not the purpose of the study to maintain the interventions after the project ends - nevertheless, we have added text at the end of the discussion to indicate what we are doing to encourage sustainability. It is quite unclear what the focus of this study isThe focus of the study is to test whether our intervention (training and support embedded within municipal action) leads to increased screening and brief advice activity, compared to no training and no embedding. We have clarified this by adding an hypothesis. It is unclear how the participating cities will be chosen and how the intervention city will be chosen. Including three different “cultures” with only one intervention and one control site for each does not seem to be a robust design.Models of international studies across differing cultures have worked well in previous international studies in this area (e.g., WHO studies and the ODHIN study). Our design and statistical measures take into account the multilevel nature of the data. We collect data on potential confounders that can be incorporated in analysis models. To clarify this further, we have added extra text in the discussions. We have added further text describing the selection of cities." } ] } ]	1	https://f1000research.com/articles/6-311

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